Aptamers to Tissue Factor Pathway Inhibitor and Their Use as Bleeding Disorder Therapeutics

ABSTRACT

The invention relates generally to the field of nucleic acids and more particularly to aptamers that bind to TFPI, which are useful as therapeutics in and diagnostics of bleeding disorders and/or other diseases or disorders in which TFPI has been implicated. In addition, the TFPI aptamers may be used before, during and/or after medical procedures to reduce complications or side effects thereof. The invention further relates to materials and methods for the administration of aptamers that bind to TFPI.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional patent application claims the benefit of priorityunder 35 U.S.C. §119(e) to U.S. Provisional Patent Application SerialNos.: 61/234,939, filed Aug. 18, 2009; 61/353,374, filed Jun. 10, 2010;61/366,362, filed Jul. 21, 2010; and 61/367,766, filed Jul. 26, 2010;the contents of each of which are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of nucleic acids and moreparticularly to aptamers that bind to tissue factor pathway inhibitor(TFPI), which are useful as therapeutics in and diagnostics of bleedingdisorders and/or other pathologies, diseases or disorders in which TFPIhas been implicated. The invention further relates to materials andmethods for the administration of aptamers that bind to TFPI.

BACKGROUND OF THE INVENTION Aptamers

An aptamer is an isolated or purified nucleic acid that binds with highspecificity and affinity to a target through interactions other thanWatson-Crick base pairing. An aptamer has a three dimensional structurethat provides chemical contacts to specifically bind to a target. Unliketraditional nucleic acid binding, aptamer binding is not dependent upona conserved linear base sequence, but rather a particular secondary ortertiary structure. That is, the nucleic acid sequences of aptamers arenon-coding sequences. Any coding potential that an aptamer may possessis entirely fortuitous and plays no role whatsoever in the binding of anaptamer to a target. A typical minimized aptamer is 5-15 kDa in size(15-45 nucleotides), binds to a target with nanomolar to sub-nanomolaraffinity, and discriminates against closely related targets (e.g.,aptamers will typically not bind to other proteins from the same gene orfunctional family).

Aptamers have been generated to many targets, such as small molecules,carbohydrates, peptides and proteins, including growth factors,transcription factors, enzymes, immunoglobulins and receptors.

Aptamers are capable of specifically binding to selected targets andmodulating the target's activity or binding interactions, e.g., throughbinding, aptamers may inhibit or stimulate a target's ability tofunction. Specific binding to a target is an inherent property of anaptamer. Functional activity, i.e., inhibiting or stimulating a target'sfunction, is not. Often times, an aptamer binds to a target and haslittle or no effect on the function of the target. Sometimes, an aptamerbinds to a target and has an inhibitory or stimulatory effect on atarget's function.

Aptamers have a number of desirable characteristics for use astherapeutics and diagnostics, including high specificity and affinity,biological activity, low immunogenicity, tunable pharmacokineticproperties and stability.

Bleeding Disorders

Coagulation is the formation of a stable fibrin/cellular hemostatic plugthat is sufficient to stop bleeding. The coagulation process, which isillustrated in FIG. 1, involves complex biochemical and cellularinteractions that can be divided into three stages. Stage 1 is theformation of activated Factor X by either the contact (intrinsic) or thetissue factor/VIIa (extrinsic) pathway. Stage 2 is the formation ofthrombin from prothrombin by Factor Xa. Stage 3 is the formation offibrin from fibrinogen stabilized by Factor XIIIa.

Hemophilia is defined as a congenital or acquired disorder ofcoagulation that usually, but not always, involves a quantitative and/orfunctional deficiency of a single coagulation protein. Deficiency ofcoagulation Factors VIII (hemophilia A) and IX (hemophilia B) are thetwo most common inherited bleeding disorders. The total overall numberof hemophilia A and B patients worldwide is approximately 400,000;however, only about ¼ (100,000) of these individuals are treated.Hemophilia A and B can be further divided in regard to the extent offactor deficiency. Mild hemophilia is 5-40% of normal factor levels andrepresents approximately 25% of the total hemophilia population.Moderate hemophilia is 1-5% of normal factor levels and representsapproximately 25% of the total hemophilia population. Severe hemophiliais <1% of normal factor levels and represents approximately 50% of thetotal hemophilia population and the highest users of currently availabletherapies.

Since the discovery of cryoprecipitation by Pool (Pool et al.,“High-potency antihaemophilic factor concentrate prepared fromcryoglobulin precipitate”, Nature, vol. 203, p. 312 (1964)), treatmentof these life threatening deficiencies has focused on factorreplacement, with a continued effort directed toward improvement in thequality of the Factor VIII and IX concentrates. The most significantimprovement has been the availability of recombinant forms of FactorsVIII and IX. These highly purified recombinant molecules have a safetyand efficacy profile that has made them the primary form of replacementfactors used for the treatment of hemophilia. The majority of mild andmoderate patients are treated “on demand”, that is when a bleed occurs.Approximately 50-60% of severe patients are treated “on demand”, whilethe remainder of this population uses prophylactic therapy, whichinvolves administering intravenous factor 2-3 times weekly.

Unfortunately, recombinant factors still retain some of the limitationsof concentrates and more highly purified plasma derived factors. Theselimitations include the relatively short half-life of the molecules,which require frequent injection to maintain effective plasmaconcentration; high cost; and the development of antibody responses,especially to Factor VIII, in a subpopulation of patients calledinhibitor patients.

In a majority of patients who develop inhibitory antibodies, theantibody is only transient. In those patients with a sustained antibodyresponse (˜15%), some respond to complex and expensive tolerizationprotocols. Those who do not respond to tolerization (˜5-10%) require theuse of non-Factor VIII/Factor IX products to control bleeding.Prothrombin Complex Concentrations (PCC), Factor Eight Inhibitor BypassAgent (FEIBA) and recombinant Factor VIIa (NovoSeven®, FVIIa) areeffective Factor VIII/Factor IX bypass treatments for inhibitorpatients.

Recombinant Factor VIIa (rFVIIa) treatment is the most used of thesebypass agents. Factor VIIa complexes with endogenous tissue factor toactivate the extrinsic pathway. It also can directly activate Factor X.The response to rFVIIa treatment is variable. The variable response,along with the poor pharmacokinetic (PK) profile of rFVIIa, can requiremultiple injections to control bleeding and significantly limits itsutility for prophylactic treatment.

A major effort is currently underway towards development of modifiedFactor VIII, IX and VIIa molecules with improved potency, stability andcirculating half-life. It should be noted that in all instances, theproducts represent incremental improvements to stability,pharmacokinetics and/or formulation of existing replacement factors.

The tissue factor/VIIa (extrinsic) pathway provides for rapid formationof low levels of thrombin that can serve as the initial hemostaticresponse to initiate and accelerate the Factor VIII, V and IX dependentintrinsic pathway. Tissue factor, Factor VIIa and Factor Xa have acentral role in this pathway and it is closely regulated by anendothelial cell associated Kunitz Type proteinase inhibitor, tissuefactor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor is a 40 kDa serine protease inhibitorthat is synthesized in and found bound to endothelial cell surfaces(“surface TFPI”), in plasma at a concentration of 2-4 nM (“plasma TFPI”)and is stored (200 pM/10⁸ platelets) and released from activatedplatelets. Approximately 10% of plasma TFPI is unassociated, while 90%is associated with oxidized LDL particles and is inactive. There are twoprimary forms of TFPI, TFPIα and TFPIβ (FIGS. 2 and 3).

TFPIα contains 3 Kunitz decoy domains, K1, K2 and K3. K1 and K2 mimicprotease substrates and inhibit by tight but reversible binding to thetarget proteases. In the case of TFPIα, K1 binds to and inhibits tissuefactor/VIIa, while K2 binds to and inhibits Factor Xa. The role for K3is unknown at this time, but it may have a role in cell-surface bindingand enhancing the inhibition of Factor Xa by K2. TFPIα has a basicC-terminal tail peptide that is the membrane binding site region for themolecule. It is estimated that 80% of the surface TFPI is TFPIα. TFPIαis primarily bound to the endothelial surface associated with themembrane proteoglycans. Heparin has been shown to release TFPIα fromcultured endothelium, isolated veins and following intravenous (IV)heparin (unfractionated and LMWH) injection. The exact nature of therelease mechanism is unclear (competition or induced release), but TFPIlevels can be increased 3-8 fold following IV heparin administration.Some TFPIα can also be found bound to glycosylated phosphatidylinositol(GPI) via an unidentified co-receptor.

TFPIβ is an alternatively spliced version of TFPI that ispost-translationally modified with a glycosylated phosphatidylinositol(GPI) anchor. It is estimated that it represents about 20% of thesurface TFPI in cultured endothelial cells. Although it has in vitroinhibitory activity, the functional in vivo role is less clear.

Surface TFPI may have a more important role in regulation of coagulationbased on its localization to the site of vascular injury and thrombusformation. Surface TFPI represents the largest proportion of activeTFPI. Data from several laboratories suggest that TFPI can also havecomplementary/synergistic effects via interactions with antithrombin III(ATIII) and protein C.

TFPI binds to Factor VIIa and Factor Xa via its K1 and K2 domains and toproteoglycans via its K3 and C-terminal domains. The fact that TFPI hasa key role in the inhibition of both tissue factor/VIIa and Xa suggeststhat TFPI inhibition could provide a single treatment or an adjuvanttreatment that is given in addition to or combined with recombinantpurified factors. An approach to promote a prothrombotic state could bevia the upregulation of the tissue factor mediated extrinsic pathway ofcoagulation. It has been suggested that inhibition of TFPI might improvecoagulation in the hemophilia patient.

Studies have demonstrated that TFPI deficiency in mice can increasethrombus formation, and that TFPI antibodies improve bleeding times inFactor VIII deficient rabbits and shorten clotting in plasma fromhemophilia patients. In the rabbit, transient hemophilia A was inducedby treating rabbits with a Factor VIII antibody. This was followed bytreatment with either Factor VIII replacement or an antibody specific torabbit TFPI. The anti-TFPI treatment produced a reduction in bleedingand a correction of coagulation that was similar to that observed withFactor VIII replacement. Liu et al. (Liu et al., “Improved coagulationin bleeding disorders by Non-Anticoagulant Sulfated Polysaccharides(NASP)”, Thromb. Haemost., vol. 95, pp. 68-76 (2006)) reported theeffects of a non-anticoagulant polysaccharide isolated from brown algaethat inhibits TFPI. A subsequent paper by Prasad et al. (Prasad et al.,“Efficacy and safety of a new-class of hemostatic drug candidate, AV513,in dogs with hemophilia A”, Blood, vol. 111, pp. 672-679 (2008)) alsoassessed this polysaccharide in hemophilia A dogs. In both studies, itwas found that TFPI inhibition had a positive effect on restoration of anormal coagulation profile and, in the dog model, an improvement inhemostatic profile, including an improved thromboelastogram (TEG) and areduction in nail bleeding time. These data suggest that inhibition ofTFPI could provide an approach to treating hemophilia.

Accordingly, it would be beneficial to identify novel therapies forantagonizing TFPI in the treatment of bleeding disorders, or that areused in conjunction with medical procedures, or that are used incombination with another drug or another therapy to induce apro-coagulant state. The present invention provides materials andmethods to meet these and other needs.

SUMMARY OF THE INVENTION

The invention provides aptamers that bind to tissue factor pathwayinhibitor (TFPI), referred to herein as “TFPI aptamers”, and methods forusing such aptamers in the treatment of bleeding disorders and otherTFPI-mediated pathologies, diseases or disorders, with or without otheragents. In addition, the TFPI aptamers may be used before, during and/orafter medical procedures, with or without other agents, in order toreduce the complications or side effects thereof.

The TFPI aptamers bind to or otherwise interact with TFPI or one or moreportions (or regions) thereof. For example, the TFPI aptamers may bindto or otherwise interact with a linear portion or a conformationalportion of TFPI. A TFPI aptamer binds to or otherwise interacts with alinear portion of TFPI when the aptamer binds to or otherwise interactswith a contiguous stretch of amino acid residues that are linked bypeptide bonds. A TFPI aptamer binds to or otherwise interacts with aconformational portion of TFPI when the aptamer binds to or otherwiseinteracts with non-contiguous amino acid residues that are broughttogether by folding or other aspects of the secondary and/or tertiarystructure of the polypeptide chain. Preferably, the TFPI is human TFPI.Preferably, the TFPI aptamers bind to TFPI and require binding contacts,at least in part, outside of the K1 and K2 regions, such as theK3/C-terminal region. More preferably, the TFPI aptamers bind at leastin part to one or more portions of mature TFPI (for example, FIG. 3A)that are selected from the group consisting of: amino acids 148-170,amino acids 150-170, amino acids 155-175, amino acids 160-180, aminoacids 165-185, amino acids 170-190, amino acids 175-195, amino acids180-200, amino acids 185-205, amino acids 190-210, amino acids 195-215,amino acids 200-220, amino acids 205-225, amino acids 210-230, aminoacids 215-235, amino acids 220-240, amino acids 225-245, amino acids230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265,amino acids 250-270, amino acids 255-275, amino acids 260-276, aminoacids 148-175, amino acids 150-175, amino acids 150-180, amino acids150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200,amino acids 150-205, amino acids 150-210, amino acids 150-215, aminoacids 150-220, amino acids 150-225, amino acids 150-230, amino acids150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250,amino acids 150-255, amino acids 150-260, amino acids 150-265, aminoacids 150-270, amino acids 150-275, amino acids 150-276, amino acids190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276,amino acids 161-181, amino acids 162-181, amino acids 182-240, aminoacids 182-241, and amino acids 182-276. Preferably, the TFPI aptamer hasa dissociation constant for TFPI of 100 nM or less.

Examples of TFPI aptamers include, but are not limited to, aptamers thatcomprise a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1, which is referred to herein as ARC26835; SEQ ID NO: 2,which is referred to herein as ARC17480; SEQ ID NO: 3, which is referredto herein as ARC 19498; SEQ ID NO: 4, which is referred to herein asARC19499; SEQ ID NO: 5, which is referred to herein as ARC19500; SEQ IDNO: 6, which is referred to herein as ARC19501; SEQ ID NO: 7, which isreferred to herein as ARC31301; SEQ ID NO: 8, which is referred toherein as ARC 18546; SEQ ID NO: 9, which is referred to herein asARC19881; and SEQ ID NO: 10, which is referred to herein as ARC19882.

Preferably, the TFPI aptamer is an aptamer or a salt thereof comprisingthe following nucleic acid sequence:mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 1) (ARC26835), wherein “dN” is a deoxynucleotide and “mN” isa 2′-O Methyl containing nucleotide (which is also known in the art as a2′-OMe, 2′-methoxy or 2′-OCH₃ containing nucleotide). In someembodiments, the TFPI aptamer is an aptamer or a salt thereof thatconsists of the nucleic acid sequence of SEQ ID NO: 1.

More preferably, the TFPI aptamer is an aptamer or a salt thereofcomprising the following nucleic acid sequence:mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2) (ARC17480), wherein “3T” is an inverted deoxythymidine,“dN” is a deoxynucleotide and “mN” is a 2′-O Methyl containingnucleotide. In some embodiments, the TFPI aptamer is an aptamer or asalt thereof that consists of the nucleic acid sequence of SEQ ID NO: 2.

Even more preferably, the TFPI aptamer is an aptamer or a salt thereofcomprising the following nucleic acid sequence:NH₂-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 3) (ARC19498), wherein “NH₂” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide. In someembodiments, the TFPI aptamer is an aptamer or a salt thereof thatconsists of the nucleic acid sequence of SEQ ID NO: 3.

Most preferably, the TFPI aptamer is an aptamer or a salt thereofcomprising the following nucleic acid sequence:PEG40K—NH-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 4) (ARC19499), wherein “NH” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide, “mN” is a 2′-O Methyl containing nucleotide and “PEG”is a polyethylene glycol. In some embodiments, the TFPI aptamer is anaptamer or a salt thereof that consists of the nucleic acid sequence ofSEQ ID NO: 4. In some embodiments, the PEG40K moiety of SEQ ID NO: 4 isa branched PEG moiety having a total molecular weight of 40 kDa. Inother embodiments, the PEG40K moiety of SEQ ID NO: 4 is a linear PEGmoiety having a molecular weight of 40 kDa. In further embodiments, thePEG40K moiety of SEQ ID NO: 4 is a methoxypolyethylene glycol (mPEG)moiety having a molecular weight of 40 kDa. In still furtherembodiments, the PEG40K moiety of SEQ ID NO: 4 is a branched mPEG moietythat contains two mPEG20K moieties, each having a molecular weight of 20kDa, as shown in FIGS. 6-9, where “20KPEG” refers to a mPEG moietyhaving a molecular weight of 20 kDa. In a preferred embodiment, thePEG40K moiety of SEQ ID NO: 4 is the branched PEG40K moiety shown inFIG. 6, where “20KPEG” refers to a mPEG moiety having a molecular weightof 20 kDa, and is connected to the aptamer as shown in FIG. 7. In a morepreferred embodiment, the PEG40K moiety is connected to the aptamerusing a 5′-amine linker phosphoramidite, as shown in FIG. 8, where“20KPEG” refers to a mPEG moiety having a molecular weight of 20 kDa. Ina most preferred embodiment, the PEG40K moiety is a mPEG moiety having atotal molecular weight of 40 kDa and is connected to the aptamer using a5′-hexylamine linker phosphoramidite, as shown in FIGS. 9A and 9B.

Alternatively, the TFPI aptamer is an aptamer or a salt thereofcomprising the following nucleic acid sequence:NH₂-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-NH₂(SEQ ID NO: 5) (ARC19500), wherein “dN” is a deoxynucleotide, “mN” is a2′-O Methyl containing nucleotide and “NH₂” is from a hexylamine linkerphosphoramidite. In some embodiments, the TFPI aptamer is an aptamer ora salt thereof that consists of the nucleic acid sequence of SEQ ID NO:5.

Preferable to the TFPI aptamer of paragraph [0029] is an aptamer or asalt thereof comprising the following nucleic acid sequence:PEG20K—NH-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-NH-PEG20K(SEQ ID NO: 6) (ARC19501), wherein “dN” is a deoxynucleotide, “mN” is a2′-O Methyl containing nucleotide, “NH” is from a hexylamine linkerphosphoramidite and “PEG” is a polyethylene glycol. In some embodiments,the TFPI aptamer is an aptamer or a salt thereof that consists of thenucleic acid sequence of SEQ ID NO: 6. In some embodiments, the PEG20Kmoieties of SEQ ID NO: 6 are branched PEG moieties. In otherembodiments, the PEG20K moieties of SEQ ID NO: 6 are linear PEGmoieties. In further embodiments, the PEG20K moieties of SEQ ID NO: 6are methoxypolyethylene glycol (mPEG) moieties having a molecular weightof 20 kDa. In still further embodiments, the PEG20K moieties of SEQ IDNO: 6 are branched mPEG moieties that contain two mPEG10K moieties eachhaving a molecular weight of 10 kDa.

Alternatively, the TFPI aptamer is an aptamer or a salt thereofcomprising the following nucleic acid sequence:mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 7) (ARC31301), wherein “dN” is a deoxynucleotide and “mN” isa 2′-O Methyl containing nucleotide. In some embodiments, the TFPIaptamer is an aptamer or a salt thereof that consists of the nucleicacid sequence of SEQ ID NO: 7.

Preferable to the TFPI aptamer of paragraph [0031] is an aptamer or asalt thereof comprising the following nucleic acid sequence:mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 8) (ARC18546), wherein “3T” is an inverted deoxythymidine,“dN” is a deoxynucleotide and “mN” is a 2′-O Methyl containingnucleotide. In some embodiments, the TFPI aptamer is an aptamer or asalt thereof that consists of the nucleic acid sequence of SEQ ID NO: 8.

More preferable to the TFPI aptamer of paragraph [0031] is an aptamer ora salt thereof comprising the following nucleic acid sequence:NH₂-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 9) (ARC19881), wherein “NH₂” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide. In someembodiments, the TFPI aptamer is an aptamer or a salt thereof thatconsists of the nucleic acid sequence of SEQ ID NO: 9.

Even more preferable to the TFPI aptamer of paragraph [0031] is anaptamer or a salt thereof comprising the following nucleic acidsequence:PEG40K—NH-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 10) (ARC19882), wherein “NH” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide, “mN” is a 2′-O Methyl containing nucleotide and “PEG”is a polyethylene glycol. In some embodiments, the TFPI aptamer is anaptamer or a salt thereof that consists of the nucleic acid sequence ofSEQ ID NO: 10. In some embodiments, the PEG40K moiety of SEQ ID NO: 10is a branched PEG moiety having a total molecular weight of 40 kDa. Inother embodiments, the PEG40K moiety of SEQ ID NO: 10 is a linear PEGmoiety having a molecular weight of 40 kDa. In further embodiments, thePEG40K moiety of SEQ ID NO: 10 is a methoxypolyethylene glycol (mPEG)moiety having a molecular weight of 40 kDa. In still furtherembodiments, the PEG40K moiety of SEQ ID NO: 10 is a branched mPEGmoiety that contains two mPEG20K moieties, each having a molecularweight of 20 kDa, as shown in FIGS. 6-9, where “20KPEG” refers to a mPEGmoiety having a molecular weight of 20 kDa. In a preferred embodiment,the PEG40K moiety of SEQ ID NO: 10 is the branched PEG40K moiety shownin FIG. 6, where “20KPEG” refers to a mPEG moiety having a molecularweight of 20 kDa, and is connected to the aptamer as shown in FIG. 7. Ina more preferred embodiment, the PEG40K moiety is connected to theaptamer using a 5′-amine linker phosphoramidite, as shown in FIG. 8,where “20KPEG” refers to a mPEG moiety having a molecular weight of 20kDa. In a most preferred embodiment, the PEG40K moiety is a mPEG moietyhaving a total molecular weight of 40 kDa and is connected to theaptamer using a 5′-hexylamine linker phosphoramidite, as shown in FIGS.9A and 9B.

Preferably, the TFPI aptamers are connected to one or more PEG moieties,with or without one or more linkers. The PEG moieties may be any type ofPEG moiety. For example, the PEG moiety may be linear, branched,multiple branched, star shaped, comb shaped or a dendrimer. In addition,the PEG moiety may have any molecular weight. Preferably, the PEG moietyhas a molecular weight ranging from 5-100 kDa in size. More preferably,the PEG moiety has a molecular weight ranging from 10-80 kDa in size.Even more preferably, the PEG moiety has a molecular weight ranging from20-60 kDa in size. Yet even more preferably, the PEG moiety has amolecular weight ranging from 30-50 kDa in size. Most preferably, thePEG moiety has a molecular weight of 40 kDa in size, also referred toherein as “40 KPEG”. The same or different PEG moieties may be connectedto a TFPI aptamer. The same or different linkers or no linkers may beused to connect the same or different PEG moieties to a TFPI aptamer.

Alternatively, the TFPI aptamers may be connected to one or more PEGalternatives (rather than to one or more PEG moieties), with or withoutone or more linkers. Examples of PEG alternatives include, but are notlimited to, polyoxazoline (POZ), PolyPEG, hydroxyethylstarch (HES) andalbumin. The PEG alternative may be any type of PEG alternative, but itshould function the same as or similar to a PEG moiety, i.e., to reducerenal filtration and increase the half-life of the TFPI aptamer in thecirculation. The same or different PEG alternatives may be connected toa TFPI aptamer. The same or different linkers or no linkers may be usedto connect the same or different PEG alternatives to a TFPI aptamer.Alternatively, a combination of PEG moieties and PEG alternatives may beconnected to a TFPI aptamer, with or without one or more of the same ordifferent linkers.

Preferably, the TFPI aptamers are connected to a PEG moiety or a PEGalternative via one or more linkers. However, the TFPI aptamers may beconnected to a PEG moiety or PEG alternative directly, without the useof a linker. The linker may be any type of molecule. Examples of linkersinclude, but are not limited to, amines, thiols and azides. The linkerscan include a phosphate group. Preferably, the linker is from a 5′-aminelinker phosphoramidite. In some embodiments, the 5′-amine linkerphosphoramidite comprises 2-18 consecutive CH₂ groups. In more preferredembodiments, the 5′-amine linker phosphoramidite comprises 2-12consecutive CH₂ groups. In even more preferred embodiments, the 5′-aminelinker phosphoramidite comprises 4-8 consecutive CH₂ groups. In mostpreferred embodiments, the 5′-amine linker phosphoramidite comprises 6consecutive CH₂ groups, i.e., is a 5′-hexylamine linker phosphoramidite.One or more of the same or different linkers or no linkers may be usedto connect one or more of the same or different PEG moieties or one ormore of the same or different PEG alternatives to a TFPI aptamer.

In preferred embodiments, an aptamer, or a salt thereof, comprising thefollowing structure is provided:

wherein HN

PO₃H is from a 5′-amine linker phosphoramidite, and the aptamer is aTFPI aptamer of the invention. Preferably, the aptamer is selected fromthe group consisting of SEQ ID NOs: 2 and 8. The 20KPEG moiety can beany PEG moiety having a molecular weight of 20 kDa. Preferably, the20KPEG moiety is a mPEG moiety having a molecular weight of 20 kDa.

In alternative preferred embodiments, an aptamer, or a salt thereof,comprising the following structure is provided:

wherein HN

PO₂H is from a 5′-amine linker phosphoramidite, and the aptamer is aTFPI aptamer of the invention. Preferably, the aptamer is selected fromthe group consisting of SEQ ID NO: 1. The 20KPEG moiety can be any PEGmoiety having a molecular weight of 20 kDa. Preferably, the 20KPEGmoiety is a mPEG moiety having a molecular weight of 20 kDa

In more preferred embodiments, an aptamer, or a salt thereof, comprisingthe following structure is provided:

wherein the aptamer is a TFPI aptamer of the invention. Preferably, theaptamer is selected from the group consisting of SEQ ID NOs: 2 and 8.The 20KPEG moiety can be any PEG moiety having a molecular weight of 20kDa. Preferably, the 20KPEG moiety is a mPEG moiety having a molecularweight of 20 kDa

In alternative more preferred embodiments, an aptamer, or a saltthereof, comprising the following structure is provided:

wherein the aptamer is a TFPI aptamer of the invention. Preferably, theaptamer is selected from the group consisting of SEQ ID NO: 1. The20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa.Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weightof 20 kDa

In most preferred embodiments, an aptamer, or a salt thereof, comprisingthe following structure is provided:

wherein “n” is about 454 ethylene oxide units (PEG=20 kDa), and theaptamer is a TFPI aptamer of the invention. “n” is about 454 ethyleneoxide units because the number of n's may vary slightly for a PEG havinga particular molecular weight. Preferably, “n” ranges from 400-500ethylene oxide units. More preferably, “n” ranges from 425-475 ethyleneoxide units. Even more preferably, “n” ranges from 440-460 ethyleneoxide units. Most preferably, “n” is 454 ethylene oxide units.Preferably, the aptamer is selected from the group consisting of SEQ IDNOs: 2 and 8.

In alternative most preferred embodiments, an aptamer, or a saltthereof, comprising the following structure is provided:

wherein “n” is about 454 ethylene oxide units (PEG=20 kDa), and theaptamer is a TFPI aptamer of the invention. “n” is about 454 ethyleneoxide units because the number of n's may vary slightly for a PEG havinga particular molecular weight. Preferably, “n” ranges from 400-500ethylene oxide units. More preferably, “n” ranges from 425-475 ethyleneoxide units. Even more preferably, “n” ranges from 440-460 ethyleneoxide units. Most preferably, “n” is 454 ethylene oxide units.Preferably, the aptamer is selected from the group consisting of SEQ IDNO: 1.

The invention also provides aptamers that have substantially the sameability to bind to TFPI as any one of the aptamers shown in SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers havesubstantially the same structure as any one of the aptamers shown in SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, theaptamers have substantially the same ability to bind to TFPI andsubstantially the same structure as any one of the aptamers shown in SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention also providesaptamers that have substantially the same ability to bind to TFPI andsubstantially the same ability to modulate a biological function of TFPIas any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,9 or 10. The invention further provides aptamers that have substantiallythe same ability to bind to TFPI and substantially the same ability tomodulate blood coagulation as any one of the aptamers shown in SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention also providesaptamers that have substantially the same structure and substantiallythe same ability to modulate a biological function of TFPI as any one ofthe aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Theinvention also provides aptamers that have substantially the samestructure and substantially the same ability to modulate bloodcoagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers havesubstantially the same ability to bind to TFPI, substantially the samestructure and substantially the same ability to modulate a biologicalfunction of TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2,3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers havesubstantially the same ability to bind to TFPI, substantially the samestructure and substantially the same ability to modulate bloodcoagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9 or 10.

The TFPI aptamers may comprise at least one chemical modification.Preferably, the modification is selected from the group consisting of: achemical substitution at a sugar position, a chemical substitution at aninternucleotide linkage and a chemical substitution at a base position.Alternatively, the modification is selected from the group consistingof: incorporation of a modified nucleotide; a 3′ cap; a 5′ cap;conjugation to a high molecular weight, non-immunogenic compound;conjugation to a lipophilic compound; incorporation of a CpG motif; andincorporation of a phosphorothioate or phosphorodithioate into thephosphate backbone. The high molecular weight, non-immunogenic compoundis preferably polyethylene glycol. In some embodiments, the polyethyleneglycol is methoxypolyethylene glycol (mPEG). The 3′ cap is preferably aninverted deoxythymidine cap.

The invention also provides aptamers that bind to TFPI and have one ormore of the following characteristics: (i) includes the primarynucleotide sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 1); (ii) includes a primary nucleotide sequence that has atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to the primary nucleotide sequence shown in SEQ IDNO: 1 or 7; (iii) has substantially the same or better ability to bindto TFPI as that of an aptamer that comprises a primary nucleotidesequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9 or 10; and/or (iv) has substantially the same or betterability to modulate or inhibit TFPI as that of an aptamer comprising aprimary nucleotide sequence selected from the group consisting of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. As used herein, the term primarynucleotide sequence refers to the 5′ to 3′ linear sequence of nucleotidebases of the nucleic acid sequence that forms an aptamer, without regardto 3′ or 5′ modifications. For example, ARC26835, ARC17480, ARC19498,ARC19499, ARC19500 and ARC19501 all have the same primary nucleotidesequence.

The invention additionally provides pharmaceutical compositionscomprising a therapeutically effective amount of a TFPI aptamer or asalt thereof, and a pharmaceutically acceptable carrier or diluent.

The invention further provides a method for treating, preventing,delaying the progression of, or ameliorating a pathology, disease ordisorder mediated by TFPI by administering to a subject the abovepharmaceutical composition. Preferably, the subject is a mammal. Morepreferably, the subject is a human. Preferably, the pathology, diseaseor disorder is selected from the group consisting of: coagulation factordeficiencies, congenital or acquired, mild or moderate or severe,including hemophilia A (Factor VIII deficiency), hemophilia B (Factor IXdeficiency) and hemophilia C (Factor XI deficiency); hemophilia A or Bwith inhibitors; other factor deficiencies (V, VII, X, XIII,prothrombin, fibrinogen); deficiency of α2-plasmin inhibitor; deficiencyof plasminogen activator inhibitor 1; multiple factor deficiency;functional factor abnormalities (e.g., dysprothrombinemia); jointhemorrhage (hemarthrosis), including, but not limited to, ankle, elbowand knee; spontaneous bleeding in other locations (muscle,gastrointestinal, mouth, etc.); hemorrhagic stroke; intracranialhemorrhage; lacerations and other hemorrhage associated with trauma;acute traumatic coagulopathy; coagulopathy associated with cancer (e.g.,acute promyelocytic leukemia); von Willebrand's Disease; disseminatedintravascular coagulation; liver disease; menorrhagia; thrombocytopeniaand hemorrhage associated with the use of anticoagulants (e.g., vitaminK antagonists, FXa antagonists, etc.).

The pharmaceutical compositions may be administered by numerous routesof administration. Preferably, the compositions are administeredintravenously (IV). Most preferably, the compositions are administeredsubcutaneously (SC or SQ).

The pharmaceutical compositions may be administered using varioustreatment regimens. For example, the compositions may be administered asa maintenance therapy at a defined dose for a defined period of time,such as when a patient is not suffering from a bleeding episode.Alternatively, the compositions may be administered on demand, i.e., asneeded, such as when a patient is suffering from a bleeding episode. Ina further alternative embodiment, the compositions may be administeredas a combination of maintenance therapy and on demand therapy. In suchan embodiment, the compositions may be administered as a maintenancetherapy at a defined dose for a defined period of time until a bleedoccurs, in which case the dosage of the compositions would be increasedon an as needed basis until the bleeding stopped, at which point thedosage of the compositions would be decreased back to the priormaintenance level. In another such embodiment, the compositions may beadministered as a maintenance therapy at a defined dose for a definedperiod of time until a bleed occurs, in which case another bleedingdisorder therapy would be administered to the patient (such as FactorVIII) until the bleeding stopped, at which point the other bleedingdisorder therapy would be discontinued. During this entire time, thecompositions would continue to be administered as a maintenance therapy.In yet another such embodiment, the compositions may be administered asa maintenance therapy at a defined dose for a defined period of timeuntil a bleed occurs, in which case the dosage of the compositions wouldbe decreased and another bleeding disorder therapy would be administeredto the patient (such as Factor VIII) until the bleeding stopped, atwhich point the dosage of the compositions would be increased back tothe prior maintenance level and the other bleeding disorder therapywould be discontinued. In another such embodiment, another bleedingdisorder therapy (such as Factor VIII) may be administered as amaintenance therapy at a defined dose for a defined period of time untila bleed occurs, in which case the compositions would be administered tothe patient until the bleeding stopped, at which point therapy with thecompositions would be discontinued. During this entire time, the otherbleeding disorder therapy would continue to be administered as amaintenance therapy. In yet another such embodiment, another bleedingdisorder therapy (such as Factor VIII) may be administered as amaintenance therapy at a defined dose for a defined period of time untila bleed occurs, in which case the dosage of the other bleeding disordertherapy would be decreased and the compositions would be administered tothe patient until the bleeding stopped, at which point the dosage of theother bleeding disorder therapy would be increased back to the priormaintenance level and therapy with the compositions would bediscontinued.

The pharmaceutical compositions may also be administered prior to,during and/or after a medical procedure. For example, the pharmaceuticalcompositions may be administered in conjunction (before, during and/orafter) with medical procedures, such as: prophylaxis and/or treatmentassociated with bleeding caused by dental procedures, orthopedic surgeryincluding but not limited to arthroplasty (e.g., hip replacement),surgical or radionuclide synovectomy (RSV), major surgery, venipuncture,transfusion and amputation.

The pharmaceutical compositions may also be administered in combinationwith another drug, such as: activated prothrombin complex concentrates(APCC), Factor Eight Inhibitor Bypass Agent (FEIBA®), recombinant FactorVIIa (e.g., NovoSeven®), recombinant Factor VIII (Advate®, Kogenate®,Recombinate®, Helixate®, ReFacto®), plasma-derived Factor VIII (HumateP®, Hemofil M®, recombinant Factor IX)(BeneFIX®, plasma-derived FactorIX (Bebulin VH®, Konyne®, Mononine®), cryoprecipitate, desmopressinacetate (DDAVP), epsilon-aminocaproic acid or tranexamic acid.Alternatively, the pharmaceutical compositions may be administered incombination with another therapy, such as: blood or blood-producttransfusion, plasmapheresis, immune tolerance induction therapy withhigh doses of replacement factor, immune tolerance therapy withimmunosuppressive agents (e.g., prednisone, rituximab) or pain therapy.

The TFPI aptamers may be used for identification of the TFPI protein.Specifically, the TFPI aptamers may be used to identify, quantify orotherwise detect the presence of the TFPI protein in a sample, such as abiological sample or other subject-derived sample. For example, the TFPIaptamers may be used in in vitro assays, e.g., ELISA, to detect TFPIlevels in a patient sample.

The invention also provides a method for regulating TFPI in which amolecule binds or otherwise interacts with one or more portions of TFPI,wherein at least one portion is outside of the K1 and K2 domains ofTFPI, such as the K3/C terminal region. The molecule can be any type ofmolecule, such as, for example, a small molecule organic compound, anantibody, a protein or peptide, a polysaccharide, a nucleic acid, ansiRNA, an aptamer, or any combination thereof. Preferably, the moleculeis a small molecule organic compound. More preferably, the molecule isan antibody. Most preferably, the molecule is an aptamer. For example,the molecule may bind to or otherwise interact with a linear portion ora conformational portion of TFPI. A molecule binds to or otherwiseinteracts with a linear portion of TFPI when the molecule binds to orotherwise interacts with a contiguous stretch of amino acid residuesthat are linked by peptide bonds. A molecule binds to or otherwiseinteracts with a conformational portion of TFPI when the molecule bindsto or otherwise interacts with non-contiguous amino acid residues thatare brought together by folding or other aspects of the secondary and/ortertiary structure of the polypeptide chain. Preferably, the moleculebinds at least in part to one or more portions of mature TFPI (forexample, FIG. 3A) that are selected from the group consisting of: aminoacids 148-170, amino acids 150-170, amino acids 155-175, amino acids160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195,amino acids 180-200, amino acids 185-205, amino acids 190-210, aminoacids 195-215, amino acids 200-220, amino acids 205-225, amino acids210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245,amino acids 230-250, amino acids 235-255, amino acids 240-260, aminoacids 245-265, amino acids 250-270, amino acids 255-275, amino acids260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180,amino acids 150-185, amino acids 150-190, amino acids 150-195, aminoacids 150-200, amino acids 150-205, amino acids 150-210, amino acids150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230,amino acids 150-235, amino acids 150-240, amino acids 150-245, aminoacids 150-250, amino acids 150-255, amino acids 150-260, amino acids150-265, amino acids 150-270, amino acids 150-275, amino acids 150-276,amino acids 190-240, amino acids 190-276, amino acids 240-276, aminoacids 242-276, amino acids 161-181, amino acids 162-181, amino acids182-240, amino acids 182-241, and amino acids 182-276. The moleculepreferably comprises a dissociation constant for human TFPI, or avariant thereof, of less than 100 μM, less than 1 μM, less than 500 nM,less than 100 nM, preferably 50 nM or less, preferably 25 nM or less,preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nMor less, even more preferably 1 nM or less, and most preferably 500 pMor less.

The invention further provides for the use of a TFPI aptamer in themanufacture of a medicament in the treatment, prevention, delayingprogression, and/or amelioration of a bleeding disorder. For example,ARC26835, ARC17480, ARC19498, ARC19499, ARC19500, ARC19501, ARC31301,ARC18546, ARC19881 and ARC19882 are used in the manufacture of amedicament for treating, preventing, delaying progression of orotherwise ameliorating a bleeding disorder.

In one embodiment, the invention provides a TFPI aptamer for use in amethod of treatment, prevention, delaying progression and/oramelioration of a bleeding disorder.

In one embodiment, the invention provides for the use of a TFPI aptamerin the manufacture of a diagnostic composition or product for use in amethod of diagnosis practiced on the human or animal body. In someembodiments, the method of diagnosis is for the diagnosis of a bleedingdisorder.

In one embodiment, the invention provides a TFPI aptamer for use in amethod of diagnosis practiced on the human or animal body. In someembodiments, the method of diagnosis is for the diagnosis of a bleedingdisorder.

In one embodiment, the invention provides the use of a TFPI aptamer fordiagnosis in vitro. In some embodiments, the in vitro use is for thediagnosis of a bleeding disorder.

The invention further relates to agents that reverse the effects of theTFPI aptamers, referred to herein as “TFPI reversal agents”. The TFPIreversal agent can be any type of molecule, such as a protein, antibody,small molecule organic compound or an oligonucleotide. Preferably, aTFPI reversal agent is an oligonucleotide that is 10-15 nucleotides inlength. Preferably, a TFPI reversal agent binds to a TFPI aptamer.Preferably, such binding is via complementary base pairing. Withoutwishing to be bound by theory, a TFPI reversal agent acts by hybridizingto a TFPI aptamer, thereby disrupting the TFPI aptamer's structure andpreventing the binding of the TFPI aptamer to TFPI.

Examples of TFPI reversal agents include, but are not limited to: SEQ IDNO: 15, which is ARC23085; SEQ ID NO: 16, which is ARC23087; SEQ ID NO:17, which is ARC23088; and SEQ ID NO: 18, which is ARC23089.

Preferably, the TFPI reversal agent is a nucleic acid comprising thestructure set forth below: mA-mG-mC-mC-mA-mA-mG-mU-mA-mU-mA-mU-mU-mC-mC(SEQ ID NO: 15), wherein “mN” is a 2′-O Methyl containing residue (whichis also known in the art as a 2′-OMe, 2′-methoxy or 2′-OCH₃ containingresidue).

Alternatively, the TFPI reversal agent is a nucleic acid comprising thestructure set forth below: mU-mA-mU-mA-mU-mA-mC-mG-mC-mA-mC-mC-mU-mA-mA(SEQ ID NO: 16), wherein “mN” is a 2′-O Methyl containing residue.

Alternatively, the TFPI reversal agent is a nucleic acid comprising thestructure set forth below: mC-mU-mA-mA-mC-mG-mA-mG-mC-mC (SEQ ID NO:17), wherein “mN” is a 2′-O Methyl containing residue.

Alternatively, the TFPI reversal agent is a nucleic acid comprising thestructure set forth below: mC-mA-mC-mC-mU-mA-mA-mC-mG-mA-mG-mC-mC-mA-mA(SEQ ID NO: 18), wherein “mN” is a 2′-O Methyl containing residue.

The invention further provides a method for treating, preventing,delaying the progression of and/or ameliorating a bleeding disorder, themethod comprising the step of administering a TFPI reversal agent to apatient in need of such treatment.

The invention provides for the use of a TFPI reversal agent in themanufacture of a medicament for the treatment, prevention, delayingprogression and/or amelioration of a bleeding disorder.

Accordingly, the invention provides for the use of a TFPI reversal agentin the manufacture of a medicament for the treatment, prevention,delaying progression and/or amelioration of a bleeding disorder in apatient wherein the method involves administering the TFPI reversalagent to the patient to control and/or modulate the therapeutic effectof a TFPI aptamer administered to the patient. The TFPI aptamer may beadministered prior to the TFPI reversal agent, simultaneously with theTFPI reversal agent or after the TFPI reversal agent, and may beadministered as part of a combination therapy. Preferably, the TFPIaptamer is administered to the patient in order to treat, prevent, delayprogression of and/or ameliorate a bleeding disorder in the patient.

The invention also provides the use of a TFPI reversal agent in themanufacture of a medicament for use in controlling and/or modulating thetreatment of a bleeding disorder, wherein the bleeding disorder is beingtreated with a TFPI aptamer.

In one embodiment, the invention provides a TFPI reversal agent for usein the treatment, prevention, delaying progression and/or ameliorationof a bleeding disorder.

Accordingly, the invention provides a TFPI reversal agent for use in thetreatment, prevention, delaying progression and/or amelioration of ableeding disorder in a patient wherein the method involves administeringthe TFPI reversal agent to the patient to control and/or modulate thetherapeutic effect of a TFPI aptamer administered to the patient.

The invention also provides a TFPI reversal agent for use in thetreatment, prevention, delaying progression and/or amelioration of ableeding disorder, wherein the bleeding disorder is being treated with aTFPI aptamer.

In one embodiment, the invention provides for the use of a TFPI reversalagent in the manufacture of a diagnostic composition or product for usein a method of diagnosis practiced on the human or animal body. In someembodiments, the method of diagnosis is for the diagnosis of a bleedingdisorder.

In one embodiment, the invention provides a TFPI reversal agent for usein a method of diagnosis practiced on the human or animal body. In someembodiments, the method of diagnosis is for the diagnosis of a bleedingdisorder.

In one embodiment, the invention provides the use of a TFPI reversalagent for diagnosis in vitro. In some embodiments, the in vitro use isfor the diagnosis of a bleeding disorder.

The invention also provides a kit comprising at least one containercomprising a quantity of one or more TFPI aptamers, along withinstructions for using the one or more TFPI aptamers in the treatment,prevention, delaying progression and/or amelioration of a bleedingdisorder. For example, the kit includes ARC26835, ARC17480, ARC19498,ARC19499, ARC19500, ARC19501, ARC31301, ARC18546, ARC19881 or ARC19882and combinations thereof. In some embodiments, the aptamers areformulated as a pharmaceutical composition. The kit may further comprisea TFPI reversal agent, along with instructions regarding administrationof the reversal agent.

The invention also provides a method for producing an aptamer that bindsto TFPI, the method comprising the step of chemically synthesizing anucleic acid having a nucleic acid sequence of an aptamer that binds toTFPI as described herein. The method may further comprise the step offormulating a pharmaceutical composition by mixing the synthesizednucleic acid sequence, or a salt thereof, with a pharmaceuticallyacceptable carrier or diluent.

The invention additionally provides a method for producing a reversalagent, the method comprising the step of chemically synthesizing anucleic acid having a nucleic acid sequence of a TFPI reversal agent asdescribed herein. The method may further comprise the step offormulating a pharmaceutical composition by mixing the synthesizednucleic acid sequence or a salt thereof, with a pharmaceuticallyacceptable carrier or diluent.

The invention further provides aptamers that have been identified by theSELEX™ process, which comprises the steps of (a) contacting a mixture ofnucleic acids with TFPI under conditions in which binding occurs; (b)partitioning unbound nucleic acids from those nucleic acids that havebound to TFPI; (c) amplifying the bound nucleic acids to yield aligand-enriched mixture of nucleic acids; and, optionally, (d)reiterating the steps of binding, partitioning and amplifying through asmany cycles as desired to obtain aptamer(s) that bind to TFPI.

The invention further provides methods for identifying aptamers thatbind at least in part to or otherwise interact with one or more portionsof TFPI, which comprise the steps of (a) contacting a mixture of nucleicacids with one or more portions of TFPI under conditions in whichbinding occurs; (b) partitioning unbound nucleic acids from thosenucleic acids that have bound to TFPI; (c) amplifying the bound nucleicacids to yield a ligand-enriched mixture of nucleic acids; and,optionally, (d) reiterating the steps of contacting, partitioning andamplifying through as many cycles as desired, to obtain aptamer(s) thatbind to a portion of TFPI. This method may also include intervening oradditional cycles with binding to full-length TFPI, followed bypartitioning and amplification. For example, the TFPI aptamers may bindto or otherwise interact with a linear portion or a conformationalportion of TFPI. A TFPI aptamer binds to or otherwise interacts with alinear portion of TFPI when the aptamer binds to or otherwise interactswith a contiguous stretch of amino acid residues that are linked bypeptide bonds. A TFPI aptamer binds to or otherwise interacts with aconformational portion of TFPI when the aptamer binds to or otherwiseinteracts with non-contiguous amino acid residues that are broughttogether by folding or other aspects of the secondary and/or tertiarystructure of the polypeptide chain. Preferably, the one or more portionsof mature TFPI (for example, FIG. 3A) are selected from the groupconsisting of: amino acids 148-170, amino acids 150-170, amino acids155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190,amino acids 175-195, amino acids 180-200, amino acids 185-205, aminoacids 190-210, amino acids 195-215, amino acids 200-220, amino acids205-225, amino acids 210-230, amino acids 215-235, amino acids 220-240,amino acids 225-245, amino acids 230-250, amino acids 235-255, aminoacids 240-260, amino acids 245-265, amino acids 250-270, amino acids255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175,amino acids 150-180, amino acids 150-185, amino acids 150-190, aminoacids 150-195, amino acids 150-200, amino acids 150-205, amino acids150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225,amino acids 150-230, amino acids 150-235, amino acids 150-240, aminoacids 150-245, amino acids 150-250, amino acids 150-255, amino acids150-260, amino acids 150-265, amino acids 150-270, amino acids 150-275,amino acids 150-276, amino acids 190-240, amino acids 190-276, aminoacids 240-276, amino acids 242-276, amino acids 161-181, amino acids162-181, amino acids 182-240, amino acids 182-241, and amino acids182-276. The aptamer preferably comprises a dissociation constant forhuman TFPI or a variant or one or more portions thereof, of less than100 μM, less than 1 μM, less than 500 nM, less than 100 nM, preferably50 nM or less, preferably 25 nM or less, preferably 10 nM or less,preferably 5 nM or less, more preferably 3 nM or less, even morepreferably 1 nM or less, and most preferably 500 pM or less.

The invention also provides methods for identifying aptamers that bindat least in part to or otherwise interact with one or more portions ofTFPI, which comprise the steps of (a) contacting a mixture of nucleicacids with full-length TFPI or one or more portions of TFPI underconditions in which binding occurs; (b) partitioning unbound nucleicacids from those nucleic acids that have bound to full-length TFPI orone or more portions of TFPI; (c) specifically eluting the bound nucleicacids with a portion of TFPI, or a ligand that binds to full-length TFPIor a portion of TFPI; (d) amplifying the bound nucleic acids to yield aligand-enriched mixture of nucleic acids; and, optionally, (e)reiterating the steps of contacting, partitioning, eluting andamplifying through as many cycles as desired to obtain aptamer(s) thatbind to one or more portions of TFPI. For example, the TFPI aptamers maybind to or otherwise interact with a linear portion or a conformationalportion of TFPI. A TFPI aptamer binds to or otherwise interacts with alinear portion of TFPI when the aptamer binds to or otherwise interactswith a contiguous stretch of amino acid residues that are linked bypeptide bonds. A TFPI aptamer binds to or otherwise interacts with aconformational portion of TFPI when the aptamer binds to or otherwiseinteracts with non-contiguous amino acid residues that are broughttogether by folding or other aspects of the secondary and/or tertiarystructure of the polypeptide chain. Preferably, the one or more portionsof mature TFPI (for example, FIG. 3A) are selected from the groupconsisting of: amino acids 148-170, amino acids 150-170, amino acids155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190,amino acids 175-195, amino acids 180-200, amino acids 185-205, aminoacids 190-210, amino acids 195-215, amino acids 200-220, amino acids205-225, amino acids 210-230, amino acids 215-235, amino acids 220-240,amino acids 225-245, amino acids 230-250, amino acids 235-255, aminoacids 240-260, amino acids 245-265, amino acids 250-270, amino acids255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175,amino acids 150-180, amino acids 150-185, amino acids 150-190, aminoacids 150-195, amino acids 150-200, amino acids 150-205, amino acids150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225,amino acids 150-230, amino acids 150-235, amino acids 150-240, aminoacids 150-245, amino acids 150-250, amino acids 150-255, amino acids150-260, amino acids 150-265, amino acids 150-270, amino acids 150-275,amino acids 150-276, amino acids 190-240, amino acids 190-276, aminoacids 240-276, amino acids 242-276, amino acids 161-181, amino acids162-181, amino acids 182-240, amino acids 182-241, and amino acids182-276. The aptamer preferably comprises a dissociation constant forhuman TFPI or a variant or one or more portions thereof of less than 100μM, less than 1 μM, less than 500 nM, less than 100 nM, preferably 50 nMor less, preferably 25 nM or less, preferably 10 nM or less, preferably5 nM or less, more preferably 3 nM or less, even more preferably 1 nM orless, and most preferably 500 pM or less.

The invention further provides methods for identifying aptamers thatbind at least in part to or otherwise interact with one or more portionsof TFPI, which comprise the steps of (a) contacting a mixture of nucleicacids with full-length TFPI or one or more portions of TFPI underconditions in which binding occurs in the presence of a TFPI ligand (aligand that binds to TFPI) that blocks one or more epitopes on TFPI fromaptamer binding; (b) partitioning unbound nucleic acids from thosenucleic acids that have bound to full-length TFPI or one or moreportions of TFPI; (c) amplifying the bound nucleic acids to yield aligand-enriched mixture of nucleic acids; and, optionally, (d)reiterating the steps of contacting, partitioning and amplifying throughas many cycles as desired to obtain aptamer(s) that bind to one or moreportions of TFPI. In other embodiments of this method, inclusion of aTFPI ligand that blocks one or more portions on TFPI from aptamerbinding can occur during the contacting step, the partitioning step, orboth. For example, the TFPI aptamers may bind to or otherwise interactwith a linear portion or a conformational portion of TFPI. A TFPIaptamer binds to or otherwise interacts with a linear portion of TFPIwhen the aptamer binds to or otherwise interacts with a contiguousstretch of amino acid residues that are linked by peptide bonds. A TFPIaptamer binds to or otherwise interacts with a conformational portion ofTFPI when the aptamer binds to or otherwise interacts withnon-contiguous amino acid residues that are brought together by foldingor other aspects of the secondary and/or tertiary structure of thepolypeptide chain. Preferably, the one or more portions of mature TFPI(for example, FIG. 3A) are selected from the group consisting of: aminoacids 148-170, amino acids 150-170, amino acids 155-175, amino acids160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195,amino acids 180-200, amino acids 185-205, amino acids 190-210, aminoacids 195-215, amino acids 200-220, amino acids 205-225, amino acids210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245,amino acids 230-250, amino acids 235-255, amino acids 240-260, aminoacids 245-265, amino acids 250-270, amino acids 255-275, amino acids260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180,amino acids 150-185, amino acids 150-190, amino acids 150-195, aminoacids 150-200, amino acids 150-205, amino acids 150-210, amino acids150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230,amino acids 150-235, amino acids 150-240, amino acids 150-245, aminoacids 150-250, amino acids 150-255, amino acids 150-260, amino acids150-265, amino acids 150-270, amino acids 150-275, amino acids 150-276,amino acids 190-240, amino acids 190-276, amino acids 240-276, aminoacids 242-276, amino acids 161-181, amino acids 162-181, amino acids182-240, amino acids 182-241, and amino acids 182-276. The aptamerpreferably comprises a dissociation constant for human TFPI or a variantor one or more portions thereof of less than 100 μM, less than 1 μM,less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably25 nM or less, preferably 10 nM or less, preferably 5 nM or less, morepreferably 3 nM or less, even more preferably 1 nM or less, and mostpreferably 500 pM or less.

The invention further provides methods for identifying aptamers thatbind at least in part to or otherwise interact with one or more portionsof TFPI, which comprise the steps of (a) contacting a mixture of nucleicacids with full-length TFPI or one or more portions of TFPI underconditions in which binding occurs; (b) partitioning unbound nucleicacids from those nucleic acids that have bound to full-length TFPI orone or more portions of TFPI; (c) partitioning bound nucleic acids thathave a desired functional property from bound nucleic acids that do nothave a desired functional property; (d) amplifying the bound nucleicacids that have a desired functional property to yield a ligand-enrichedmixture of nucleic acids; and, optionally, (e) reiterating the steps ofcontacting, partitioning, partitioning and amplifying through as manycycles as desired to obtain aptamer(s) that bind to one or more portionsof TFPI. Steps (b) and (c) can occur sequentially or simultaneously.Preferably, the desired functional property is inhibition of TFPI'sinteraction with FXa, FVIIa, TFPI receptor or the glycocalyx. Forexample, the TFPI aptamers may bind to or otherwise interact with alinear portion or a conformational portion of TFPI. A TFPI aptamer bindsto or otherwise interacts with a linear portion of TFPI when the aptamerbinds to or otherwise interacts with a contiguous stretch of amino acidresidues that are linked by peptide bonds. A TFPI aptamer binds to orotherwise interacts with a conformational portion of TFPI when theaptamer binds to or otherwise interacts with non-contiguous amino acidresidues that are brought together by folding or other aspects of thesecondary and/or tertiary structure of the polypeptide chain.Preferably, the one or more portions of mature TFPI (for example, FIG.3A) are selected from the group consisting of: amino acids 148-170,amino acids 150-170, amino acids 155-175, amino acids 160-180, aminoacids 165-185, amino acids 170-190, amino acids 175-195, amino acids180-200, amino acids 185-205, amino acids 190-210, amino acids 195-215,amino acids 200-220, amino acids 205-225, amino acids 210-230, aminoacids 215-235, amino acids 220-240, amino acids 225-245, amino acids230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265,amino acids 250-270, amino acids 255-275, amino acids 260-276, aminoacids 148-175, amino acids 150-175, amino acids 150-180, amino acids150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200,amino acids 150-205, amino acids 150-210, amino acids 150-215, aminoacids 150-220, amino acids 150-225, amino acids 150-230, amino acids150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250,amino acids 150-255, amino acids 150-260, amino acids 150-265, aminoacids 150-270, amino acids 150-275, amino acids 150-276, amino acids190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276,amino acids 161-181, amino acids 162-181, amino acids 182-240, aminoacids 182-241, and amino acids 182-276. The aptamer preferably comprisesa dissociation constant for human TFPI or a variant or one or moreportions thereof of less than 100 μM, less than 1 μM, less than 500 nM,less than 100 nM, preferably 50 nM or less, preferably 25 nM or less,preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nMor less, even more preferably 1 nM or less, and most preferably 500 pMor less.

The invention also provides an aptamer that binds to a human tissuefactor pathway inhibitor (TFPI) polypeptide having the amino acidsequence of SEQ ID NO: 11, wherein the aptamer modulates TFPI-mediatedinhibition of blood coagulation, and wherein the aptamer competes forbinding to TFPI with a reference aptamer comprising a nucleic acidsequence selected from the group consisting of: SEQ ID NO: 4 (ARC19499),SEQ ID NO: 1 (ARC26835), SEQ ID NO: 2 (ARC17480), SEQ ID NO: 3(ARC19498), SEQ ID NO: 5 (ARC19500), SEQ ID NO:6 (ARC19501), SEQ ID NO:7 (ARC31301), SEQ ID NO: 8 (ARC18546), SEQ ID NO: 9 (ARC19881) and SEQID NO: 10 (ARC19882). Preferably, the reference aptamer comprises thenucleic acid sequence of SEQ ID NO: 4 (ARC 19499).

The invention further provides an aptamer that binds to a human tissuefactor pathway inhibitor (TFPI) polypeptide having the amino acidsequence of SEQ ID NO: 11, wherein the aptamer binds to a linear portionor a conformational portion of TFPI in which at least a portion of theregion recognized by the aptamer is different than the TFPI region boundby Factor VIIa, Factor Xa, or both Factor VIIa and Factor Xa.Preferably, the aptamer binds to one or more regions comprising at leasta portion of the amino acid sequence of SEQ ID NO: 11 selected from thegroup consisting of: amino acid residues 148-170, amino acid residues150-170, amino acid residues 155-175, amino acid residues 160-180, aminoacid residues 165-185, amino acid residues 170-190, amino acid residues175-195, amino acid residues 180-200, amino acid residues 185-205, aminoacid residues 190-210, amino acid residues 195-215, amino acid residues200-220, amino acid residues 205-225, amino acid residues 210-230, aminoacid residues 215-235, amino acid residues 220-240, amino acid residues225-245, amino acid residues 230-250, amino acid residues 235-255, aminoacid residues 240-260, amino acid residues 245-265, amino acid residues250-270, amino acid residues 255-275, amino acid residues 260-276, aminoacid residues 148-175, amino acid residues 150-175, amino acid residues150-180, amino acid residues 150-185, amino acid residues 150-190, aminoacid residues 150-195, amino acid residues 150-200, amino acid residues150-205, amino acid residues 150-210, amino acid residues 150-215, aminoacid residues 150-220, amino acid residues 150-225, amino acid residues150-230, amino acid residues 150-235, amino acid residues 150-240, aminoacid residues 150-245, amino acid residues 150-250, amino acid residues150-255, amino acid residues 150-260, amino acid residues 150-265, aminoacid residues 150-270, amino acid residues 150-275, amino acid residues150-276, amino acid residues 190-240, amino acid residues 190-276, aminoacid residues 240-276, amino acid residues 242-276, amino acid residues161-181, amino acid residues 162-181, amino acid residues 182-240, aminoacid residues 182-241, and amino acid residues 182-276. More preferably,the aptamer competes with a reference aptamer comprising the nucleicacid sequence of SEQ ID NO: 4 (ARC 19499) for binding to TFPI.

The invention also provides an aptamer that binds to the same region ona human tissue factor pathway inhibitor (TFPI) polypeptide having theamino acid sequence of SEQ ID NO: 11 as the region bound by a TFPIaptamer comprising the nucleic acid sequence of SEQ ID NO: 4 (ARC19499).

The invention further provides an aptamer that binds to a region on ahuman tissue factor pathway inhibitor (TFPI) polypeptide comprising oneor more portions of SEQ ID NO: 11, wherein the one or more portions isselected from the group consisting of: amino acid residues 148-170,amino acid residues 150-170, amino acid residues 155-175, amino acidresidues 160-180, amino acid residues 165-185, amino acid residues170-190, amino acid residues 175-195, amino acid residues 180-200, aminoacid residues 185-205, amino acid residues 190-210, amino acid residues195-215, amino acid residues 200-220, amino acid residues 205-225, aminoacid residues 210-230, amino acid residues 215-235, amino acid residues220-240, amino acid residues 225-245, amino acid residues 230-250, aminoacid residues 235-255, amino acid residues 240-260, amino acid residues245-265, amino acid residues 250-270, amino acid residues 255-275, aminoacid residues 260-276, amino acid residues 148-175, amino acid residues150-175, amino acid residues 150-180, amino acid residues 150-185, aminoacid residues 150-190, amino acid residues 150-195, amino acid residues150-200, amino acid residues 150-205, amino acid residues 150-210, aminoacid residues 150-215, amino acid residues 150-220, amino acid residues150-225, amino acid residues 150-230, amino acid residues 150-235, aminoacid residues 150-240, amino acid residues 150-245, amino acid residues150-250, amino acid residues 150-255, amino acid residues 150-260, aminoacid residues 150-265, amino acid residues 150-270, amino acid residues150-275, amino acid residues 150-276, amino acid residues 190-240, aminoacid residues 190-276, amino acid residues 240-276, amino acid residues242-276, amino acid residues 161-181, amino acid residues 162-181, aminoacid residues 182-240, amino acid residues 182-241, and amino acidresidues 182-276.

The invention additionally provides an aptamer that binds to humantissue factor pathway inhibitor (TFPI) and exhibits one or more of thefollowing properties: a) competes for binding to TFPI with any one ofSEQ ID NOs: 1-10; b) inhibits TFPI inhibition of Factor Xa; c) increasesthrombin generation in hemophilia plasma; d) inhibits TFPI inhibition ofthe intrinsic tenase complex; e) restores normal hemostasis, as measuredby thromboelastography (TEG®) in whole blood and plasma; f) restoresnormal clotting, as indicated by shorter clot time, more rapid clotformation or more stable clot development, as measured bythromboelastography (TEG®) or rotational thromboelastometry (ROTEM) inwhole blood and plasma; or g) decreases the clot time, as measured bydilute prothrombin time (dPT), tissue factor activated clotting time(TF-ACT) or any other TFPI-sensitive clot-time measurement.

The invention also provides an aptamer that binds to human tissue factorpathway inhibitor wherein the aptamer competes for binding to TFPI witha reference aptamer selected from the group consisting of: SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

The invention further provides an aptamer that binds to tissue factorpathway inhibitor (TFPI) wherein the aptamer competes, either directlyor indirectly, for binding to TFPI with a reference antibody selectedfrom the group consisting of: AD4903.

The invention also provides an aptamer that binds to human tissue factorpathway inhibitor (TFPI) and comprises a stem and loop motif having thenucleotide sequence of SEQ ID NO: 4, wherein: a) any one or more ofnucleotides 1, 2, 3, 4, 6, 8, 11, 12, 13, 17, 20, 21, 22, 24, 28, 30 and32 may be modified from a 2′-OMe substitution to a 2′-deoxysubstitution; b) any one or more of nucleotides 5, 7, 15, 19, 23, 27, 29and 31 may be modified from a 2′-OMe uracil to either a 2′-deoxy uracilor a 2′-deoxy thymine; c) nucleotide 18 may be modified from a 2′-OMeuracil to a 2′-deoxy uracil; and/or d) any one or more of nucleotides14, 16 and 25 may be modified from a 2′-deoxy cytosine to either a2′-OMe cytosine or a 2′-fluoro cytosine.

The invention additionally provides an aptamer that binds to humantissue factor pathway inhibitor (TFPI) and comprises nucleotides 7-28 ofSEQ ID NO: 2.

The invention further provides a method for treating a bleeding disordercomprising administering any one of the above aptamers.

The invention further provides an aptamer that binds to tissue factorpathway inhibitor (TFPI), wherein the aptamer comprises a primarynucleic acid sequence selected from the group consisting of SEQ ID NOs.:4, 1, 2, 3, 5, 6, 7, 8, 9 and 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the coagulation cascade.

FIG. 2 is an illustration of the forms of TFPI, which are associatedwith the vascular endothelium or in the plasma pool.

FIG. 3 is a schematic representation of the two forms of TFPI found onthe endothelium, (FIG. 3A) TFPIα and (FIG. 3B) TFPIβ.

FIG. 4 is a schematic representation of the in vitro aptamer selection(SELEX™) process from pools of random sequence oligonucleotides.

FIG. 5 is an illustration of the amino acid sequence of the mature humanTFPI protein.

FIG. 6 is an illustration of a 40 kDa branched PEG.

FIG. 7 is an illustration of a 40 kDa branched PEG that is attached tothe 5′ terminus of an amine aptamer.

FIG. 8 is an illustration of a 40 kDa branched PEG that is attached tothe 5′ terminus of an aptamer using a 5′-amine linker phosphoramidite.

FIG. 9A is an illustration of a 40 kDa branched PEG that is attached tothe 5′ terminus of an aptamer using a 5′-hexylamine linkerphosphoramidite. FIG. 9B is an alternative illustration of a 40 kDabranched PEG that is attached to the 5′ terminus of an aptamer using a5′-hexylamine linker phosphoramidite.

FIG. 10A is an illustration of a TFPI aptamer, which is comprised of2′-O Methyl (circles) and 2′-deoxy (squares) nucleotides and is modifiedat its 5′-terminus with a 40 kDa PEG moiety and at its 3′-terminus withan inverted deoxythymidine residue (3T, which is also known in the artas idT). FIG. 10B is an illustration of a TFPI aptamer, which iscomprised of 2′-O Methyl (circles) and 2′-deoxy (squares) nucleotidesand is modified at its 5′-terminus with a 40 kDa PEG moiety and linker,and at its 3′-terminus with an inverted deoxythymidine residue (3T).FIG. 10C is an illustration of the putative structure of ARC19499, whichis comprised of 2′-O Methyl (circles) and 2′-deoxy (squares) nucleotidesand is modified at its 5′-terminus with a 40 kDa branched PEG moiety anda hexylamine phosphate-containing linker, and at its 3′-terminus with aninverted deoxythymidine residue (3T).

FIG. 11 is an illustration depicting various PEGylation strategies, suchas standard mono-PEGylation, multiple PEGylation and oligomerization viaPEGylation.

FIG. 12A is a graph showing that ARC17480 binds tightly to full-lengthTFPI. The data are fit to both monophasic and biphasic models todetermine a K_(D) for binding. FIG. 12B is a graph showing that tRNAshifts the affinity of ARC17480 for TFPI. The aptamer still bindstightly to TFPI in the presence of tRNA, indicating that the binding ofARC17480 to TFPI is specific.

FIG. 13 depicts the results of binding-competition experiments withradiolabeled ARC17480, full-length TFPI and various unlabeled aptamers.Unlabeled ARC17480 and ARC19499 (FIG. 13A); ARC19498 (FIG. 13B),ARC18546 (FIG. 13C); ARC26835 and ARC31301 (FIG. 13D); ARC19500,ARC19501, ARC19881 and ARC19882 (FIG. 13E) all compete for binding withradiolabeled ARC17480.

FIG. 14 is a set of graphs showing binding experiments with ARC17480 andvarious proteins, including coagulation factors, protease inhibitors andcoagulation zymogens. FIG. 14A is a graph of a binding experiment withARC17480 and various proteins. FIG. 14B is a graph of a bindingexperiment with ARC17480 and TFPI or various activated coagulationfactors. FIG. 14C is a graph of a binding experiment with ARC17480 andTFPI or various protease inhibitors. FIG. 14D is a graph of a bindingexperiment with ARC17480 and TFPI or various coagulation zymogens.ARC17480 showed significant binding to TFPI, but not to any of the otherproteins tested.

FIG. 15 is a graph showing data from a plate-based assay demonstratingbinding of ARC19499 to recombinant TFPI.

FIG. 16 is a graph showing data from a plate-based competition assaydemonstrating binding of ARC19498 to TFPI in competition with ARC19499.

FIG. 17A depicts the results of a binding assay with radiolabeledARC17480, full-length TFPI and TFPI-His. FIG. 17B depicts the results ofa binding assay with radiolabeled ARC17480, full-length TFPI, truncatedTFPI-K1K2, TFPI K3-C-terminal domain protein, and the peptide thatcontains the C-terminal region of TFPI in the presence of neutravidin.

FIG. 18A depicts the results of a binding assay with radiolabeledARC17480 and full-length TFPI in the absence or presence of 0.1 mg/mLheparin. FIG. 18B depicts the results of a binding-competition assaywith radiolabeled ARC17480, 12.5 nM full-length TFPI, and differentconcentrations of heparin and low molecular weight heparin (LMWH) ascompetitors.

FIG. 19, A and B, illustrates competition of various anti-TFPIantibodies with ARC19499 in a plate-based binding assay.

FIG. 20, A, B and C, illustrates competition of various anti-TFPIantibodies with ARC19499 in a nitrocellulose filtration (dot-blot)assay.

FIG. 21 is a series of graphs showing the activity of ARC19499 in theextrinsic Xase inhibition assay. In FIG. 21A, the rate (mOD/min) wasplotted vs time (minutes). In the absence of TFPI, the rate was linear.1 nM TFPI decreased the rate dramatically. Increasing concentrations ofARC19499 from 0.01 to 1000 nM increased the rate in a dose-dependentmanner until nearly the level of no TFPI. In FIG. 21B, the rates at the4 minute time point were normalized to the rate in the absence of TFPIat 4 minutes. ARC19499 showed a dose-dependent improvement on the rateof the assay, reaching levels close to that of no TFPI by 10 nM aptamer.Data for FIG. 21A were representative from three experiments. Data forFIG. 21B represent mean±standard error, n=3.

FIG. 22, A-C, depicts the results of a Factor Xa (FXa) activity assaywith full-length TFPI and ARC17480, ARC18546, ARC26835, ARC31301,ARC19498, ARC19499, ARC19500, ARC19501, ARC19881 or ARC19882. Theadjusted rate of FXa substrate degradation is plotted as a function ofaptamer concentration. The rates are adjusted by subtraction of the rateobserved with FXa and TFPI in the absence of aptamer. All of theaptamers inhibit TFPI, which results in a concentration-dependentincrease in FXa activity in this assay.

FIG. 23 is a graph that shows protection of Factor Xa (FXa) activity byARC19499 from TFPI inhibition in a chromogenic FXa activity assay.

FIG. 24 is a graph that shows protection of the extrinsic FXase byARC19499 from TFPI inhibition in a chromogenic assay of Factor X (FX)activation.

FIG. 25 is a graph that shows protection of the TF:FVIIa complex byARC19499 from TFPI inhibition in a fluorogenic assay of TF:FVIIaactivity.

FIG. 26 is a graph that shows the effect of ARC19499 on tissue factor(TF)-initiated thrombin generation in a Normal Synthetic CoagulationProteome (SCP).

FIG. 27 is a graph that shows the effect of ARC19499 on tissue factor(TF)-initiated thrombin generation in a hemophilia A SyntheticCoagulation Proteome.

FIG. 28 is a graph that shows the effect of ARC19499 on tissue factor(TF)-initiated thrombin generation in a hemophilia B SyntheticCoagulation Proteome.

FIG. 29 is a graph that shows the effect of increasing Factor VIII(FVIII) concentrations on tissue factor (TF)-initiated thrombingeneration in the absence of ARC19499.

FIG. 30 is a graph that shows the effect of increasing Factor VIII(FVIII) concentrations on tissue factor (TF)-initiated thrombingeneration in the presence of 1.0 nM ARC19499.

FIG. 31 is a graph that shows the effect of increasing Factor VIII(FVIII) concentrations on tissue factor (TF)-initiated thrombingeneration in the presence of 2.5 nM ARC19499.

FIG. 32 is a graph that shows the effect of increasing ARC19499concentrations on tissue factor (TF)-initiated thrombin generation inthe absence of Factor VIII (FVIII).

FIG. 33 is a graph that shows the effect of increasing ARC19499concentrations on tissue factor (TF)-initiated thrombin generation inthe presence of 100% Factor VIII (FVIII).

FIG. 34 is a graph that shows the effect of increasing ARC19499concentrations on tissue factor (TF)-initiated thrombin generation inthe presence of 2% Factor VIII (FVIII).

FIG. 35 is a graph that shows the effect of increasing ARC19499concentrations on tissue factor (TF)-initiated thrombin generation inthe presence of 5% Factor VIII (FVIII).

FIG. 36 is a graph that shows the effect of increasing ARC19499concentrations on tissue factor (TF)-initiated thrombin generation inthe presence of 40% Factor VIII (FVIII).

FIG. 37 is a series of graphs showing the activity of ARC19499 in thecalibrated automated thrombogram (CAT) assay in pooled normal plasma(PNP) initiated with 0.1 pM tissue factor (TF; FIG. 37A) or 1.0 pM TF(FIG. 37B). The endogenous thrombin potential (ETP; FIG. 37C) and peakthrombin (FIG. 37D) both showed a dose-dependent increase withincreasing concentrations of ARC19499 at both TF concentrations. The lagtime (FIG. 37E) showed a dose-dependent decrease with increasingconcentrations of ARC19499 at both TF concentrations.

FIG. 38 is a series of graphs showing the activity of ARC19499 in thecalibrated automated thrombogram (CAT) assay in TFPI-depleted plasmainitiated with 0.01, 0.1 or 1.0 pM tissue factor (TF). FIG. 38A showsthrombin generation curves at increasing ARC19499 concentrations withthree different TF concentrations. The endogenous thrombin potential(ETP; FIG. 38B), peak thrombin (FIG. 38C) and lag time (FIG. 38D) showedlittle or no change over all tested ARC19499 concentrations at alltested TF concentrations.

FIG. 39 is a series of graphs showing the activity of ARC19499 in thecalibrated automated thrombogram (CAT) assay in pooled normal plasma(PNP) previously treated with a neutralizing, polyclonal anti-TFPIantibody. The assay was initiated with 0.01 pM tissue factor (TF; FIG.39A), 0.1 pM TF (FIG. 39B) or 1.0 pM TF (FIG. 39C). The endogenousthrombin potential (ETP; FIG. 39D), peak thrombin (FIG. 39E) and lagtime (FIG. 39F) remained largely unchanged at all ARC19499concentrations independent of the TF concentration.

FIG. 40 is a series of graphs showing a calibrated automated thrombogram(CAT) assay with ARC17480 (FIG. 40A), ARC19498 (FIG. 40B) and ARC19499(FIG. 40C) at various concentrations. The endogenous thrombin potential(ETP; FIG. 40D) and peak thrombin (FIG. 40E) measured with variousconcentrations of ARC17480, ARC19498 and ARC19499 in hemophilia A plasmawere similar to one another, with ARC19499 having slightly greateractivity, reaching an ETP plateau close to normal plasma levels by 30 nMaptamer. The thrombin generation curves (FIG. 40A-C) are representativedata. The ETP (FIG. 40D) and peak thrombin (FIG. 40E) data represent themean±standard error, n=3.

FIG. 41 is a graph of thrombin generation in platelet-poor normal plasmafrom a single, healthy volunteer. The plasma was treated with ananti-FVIII antibody to generate a hemophilia A-like state. ARC19499showed a dose-dependent increase in thrombin generation in theantibody-treated plasma.

FIG. 42 is a series of graphs showing a calibrated automated thrombogram(CAT) assay with ARC19499 (FIG. 42A) and ARC17480 (FIG. 42B) at variousconcentrations in hemophilia B plasma.

FIG. 43 is a series of graphs showing the effect of ARC19499 (diamonds)and ARC17480 (triangles) on endogenous thrombin potential (ETP), peakthrombin and lag time in hemophilia B plasma. The solid line designatesthe level of each parameter in the absence of any drug. The hatched linedesignates the level of each parameter in pooled normal plasma (PNP)without any additional drug. Data represent mean±standard error, n=3.Both aptamers behaved very similarly to each other in hemophilia Bplasma.

FIG. 44 is a series of graphs showing the effects of ARC19499 comparedto a negative control aptamer on thrombin generation as measured by thecalibrated automated thrombogram (CAT) assay in plasmas from patientswith hemophilia A (FIG. 44A), hemophilia A with inhibitors (FIG. 44B) orhemophilia B (FIG. 44C). The results are given in terms of the lag time(left), endogenous thrombin potential (ETP) (middle) and peak thrombinconcentration (right). In all graphs, lines represent activity of normalplasma (solid) and factor-deficient plasma (dashed) in the absence ofaptamer, and shading around the lines represents the standard error ofthe mean.

FIG. 45 depicts the results of thrombin generation experiments withARC17480, ARC18546, ARC26835 and ARC31301 in hemophilia A plasma.Adjusted endogenous thrombin potential (ETP; FIGS. 45A and C) andadjusted peak thrombin (FIGS. 45B and D) values are plotted as afunction of aptamer concentration. The ETP and peak thrombin values forhemophilia plasma were subtracted from each value to give the adjustedvalues. ARC17480, ARC18546, ARC26835 and ARC31301 increase thrombingeneration in a concentration-dependent manner in hemophilia A plasma.

FIG. 46 depicts the results of thrombin generation experiments withARC17480, ARC19500, ARC19501, ARC19881 and ARC19882 in hemophilia Aplasma. Adjusted endogenous thrombin potential (ETP; FIG. 46A) andadjusted peak thrombin (FIG. 46B) values are plotted as a function ofaptamer concentration. The ETP and peak thrombin values for hemophiliaplasma were subtracted from each value to give the adjusted values.ARC17480, ARC19500, ARC19501, ARC19881 and ARC19882 increase thrombingeneration in a concentration-dependent manner in hemophilia A plasma.

FIG. 47 is a series of graphs from thrombin generation experimentsshowing the effect of NovoSeven® (empty triangles) and ARC19499 (filleddiamonds) on endogenous thrombin potential (ETP; FIG. 47A), peakthrombin (FIG. 47B) and lag time (FIG. 47C) in normal plasma. The solidblack line designates the level of each parameter in the absence of anydrug. Data represent mean±standard error, n=3.

FIG. 48 is a series of graphs from thrombin generation experimentsshowing the effect of NovoSeven® (empty triangles) and ARC19499 (filleddiamonds) on endogenous thrombin potential (ETP; FIG. 48A), peakthrombin (FIG. 48B) and lag time (FIG. 48C) in hemophilia A plasma. Thesolid black line designates the level of each parameter in the absenceof any drug. The dashed line designates the level of each parameter inpooled normal plasma (PNP) without any additional drug. Data representmean±standard error, n=3.

FIG. 49 is a series of graphs from thrombin generation experimentsshowing the effect of NovoSeven® (empty triangles) and ARC19499 (filleddiamonds) on endogenous thrombin potential (ETP; FIG. 49A), peakthrombin (FIG. 49B) and lag time (FIG. 49C) in hemophilia A inhibitorplasma. The solid black line designates the level of each parameter inthe absence of any drug. The dashed line designates the level of eachparameter in pooled normal plasma (PNP) without any additional drug.Data represent mean±standard error, n=3.

FIG. 50 is a series of graphs from experiments showing the effect ofNovoSeven® (empty triangles) and ARC19499 (filled diamonds) on R-value(FIG. 50A), angle (FIG. 50B) and maximum amplitude (MA; FIG. 50C) in athromboelastography (TEG®) assay in citrated whole blood from healthyvolunteers. The solid black line designates the level of each parameterin the absence of any drug. Data represent mean±standard error, n=3.

FIG. 51 is a series of graphs from experiments showing the effect ofNovoSeven® (empty triangles) and ARC19499 (filled diamonds) on R-value(FIG. 51A), angle (FIG. 51B) and maximum amplitude (MA; FIG. 51C) in athromboelastography (TEG®) assay in citrated whole blood from healthyvolunteers treated with an anti-FVIII antibody. The solid black linedesignates the level of each parameter in the absence of any drug. Thedashed line designates the level of each parameter in whole blood nottreated with antibody. Data represent mean±standard error, n=3.

FIG. 52 is a series of graphs from thromboelastography experimentsshowing the lag time (FIG. 52A), peak thrombin (FIG. 52B) and endogenousthrombin potential (ETP; FIG. 52C). Each line represents the doseresponse of ARC19499 in the presence of a different percent of FactorVIII (filled diamonds, 0%; empty triangles, 1.4%; filled squares, 2.5%;filled triangles, 5%; empty squares, 14%; and filled circles, 140%). Thedashed line designates level of each parameter in the presence of poolednormal plasma (PNP) alone. The solid line designates the level of eachparameter in hemophilia A plasma without any additions. Data representmean±standard error, n=3.

FIG. 53 is a series of graphs from thrombin generation experimentsdemonstrating ARC19499 activity in plasma with various concentrations ofFactor VIII (FVIII). In FIG. 53A, the endogenous thrombin potential(ETP) is plotted as a function of ARC19499 concentration. The dashedlines represent the ETP after addition of different amounts of FVIII tohemophilia A plasma. The solid lines show that ARC19499 increases ETP inhemophilia A plasma (line with triangles) and hemophilia A plasma with5% FVIII added (line with diamonds). In FIG. 53B, the ETP is plottedversus FVIII concentration. ETP data is shown with and without theaddition of 300 nM ARC19499.

FIG. 54 illustrates the experimental design of the spatial clottingmodel. FIG. 54A is a diagram of the spatial clotting chamber. FIG. 54Bis a schematic illustration of the components of the system formeasuring clot progression in the chamber.

FIG. 55 shows two graphs illustrating clot propagation in the spatialclotting model, as measured by light scattering, plotted as a functionof distance from the activating surface. Clotting was activated by lowdensity tissue factor in normal pooled plasma in the absence (FIG. 55A)and in the presence (FIG. 55B) of 300 nM ARC19499.

FIG. 56 is a graph of clot size versus time, in the absence (thick blackline) and the presence (thin grey line) of 300 nM ARC19499 in normalpooled plasma. The parameters that can be derived from this graphinclude the lag time (time until beginning of clot growth), initialvelocity (α or V_(initial); mean slope during the first 10 minutes ofgrowth), stationary velocity (β or V_(stationary); mean slope during thenext 30 minutes of growth) and clot size after 60 minutes (an integralparameter of clot formation efficiency).

FIG. 57 is a series of graphs showing the lag time (FIG. 57A),V_(initial) (FIG. 57B), V_(stationary) (FIG. 57C) and clot size after 60minutes (FIG. 57D) in normal pooled plasma, each plotted as a functionof tissue factor density in the presence (circles) and absence (squares)of ARC19499.

FIG. 58 is a series of graphs showing the lag time (FIG. 58A),V_(initial) (FIG. 58B), V_(stationary) (FIG. 58C) and clot size after 60minutes (FIG. 58D) in normal pooled plasma, each plotted as a functionof ARC19499 concentration under conditions of low surface tissue factordensity.

FIG. 59 is a series of graphs illustrating the effect of ARC19499 on thelag time (FIG. 59A), V_(initial) (FIG. 59B), V_(stationary) (FIG. 59C)and clot size after 60 minutes (FIG. 59D) in normal pooled plasma underconditions of low surface tissue factor density. An asterisk indicates astatistically significant difference±ARC19499 (P<0.05).

FIG. 60 is a series of graphs showing the lag time (FIG. 60A),V_(initial) (FIG. 60B), V_(stationary) (FIG. 60C) and clot size after 60minutes (FIG. 60D) in normal pooled plasma, each plotted as a functionof ARC19499 concentration under conditions of medium surface tissuefactor density.

FIG. 61 is a series of graphs illustrating the effect of ARC19499 on thelag time (FIG. 61A), V_(initial) (FIG. 61B), V_(stationary) (FIG. 61C)and clot size after 60 minutes (FIG. 61D) in normal pooled plasma underconditions of medium surface tissue factor density. An asteriskindicates a statistically significant difference±ARC19499 (P<0.05).

FIG. 62 compares clot propagation in normal pooled plasma (FIG. 62A) tonormal pooled plasma containing 100 nM ARC19499 (FIG. 62B) or 100 nMrecombinant factor VIIa (rVIIa or Novoseven®; FIG. 62C) under conditionsof low surface tissue factor density.

FIG. 63 is an illustration showing a series of light scattering imagesfrom the spatial clotting model. Each row depicts clot propagation froma surface (bottom) over time 0, 10, 20, 30, 40, 50 and 60 minutes. Thetop row shows clot propagation in severe hemophilia A plasma, followedby severe hemophilia A plasma containing 100 nM ARC19499 in the secondrow and severe hemophilia A plasma containing 100 nM recombinant factorVIIa (rVIIa) in the third row.

FIG. 64 is a graph of clot size versus time, in normal plasma (darkgrey, dashed line), severe hemophilia A plasma (black, solid line),severe hemophilia A plasma containing 100 nM ARC19499 (light grey, solidline) or 100 nM recombinant factor VIIa (rVIIa) (light grey, dashedline).

FIG. 65 is a table summarizing the demographics of hemophilia A patientsfrom which plasma samples were drawn for spatial clot formationexperiments.

FIG. 66 shows the effects of ARC19499 or recombinant factor VIIa(rVIIa), titrated into severe hemophilia A plasma from Patient 1, onspatial clot formation activated with low surface tissue factor density.The effects of ARC19499 and rVIIa on lag time are depicted in FIGS. 66Aand B, respectively, while the effects of ARC19499 and rVIIa onV_(initial) are depicted in FIGS. 66C and D, respectively.

FIG. 67 shows the effects of ARC19499 or recombinant factor VIIa(rVIIa), titrated into severe hemophilia A plasma from Patient 2, onspatial clot formation activated with low surface tissue factor density.The effects of ARC19499 and rVIIa on lag time are depicted in FIGS. 67Aand B, respectively, while the effects of ARC19499 and rVIIa onV_(initial) are depicted in FIGS. 67C and D, respectively.

FIG. 68 shows the effects of ARC19499 or recombinant factor VIIa(rVIIa), titrated into severe hemophilia A plasma from Patient 3, onspatial clot formation activated with low surface tissue factor density.The effects of ARC19499 and rVIIa on lag time are depicted in FIGS. 68Aand B, respectively, while the effects of ARC19499 and rVIIa onV_(initial) are depicted in FIGS. 68C and D, respectively.

FIG. 69 shows the effects of ARC19499 (black symbols) or recombinantfactor VIIa (rVIIa; grey symbols) on V_(stationary) in hemophilia Aplasma samples from Patients 1-3, activated with low surface tissuefactor density.

FIG. 70 shows the effects of ARC19499 (FIG. 70A) or recombinant factorVIIa (rVIIa; FIG. 70B) on clot size at 60 minutes in hemophilia A plasmasamples from Patients 1-3, activated with low surface tissue factordensity.

FIG. 71 is a series of graphs illustrating the effect of 300 nM ARC19499on the average lag time (FIG. 71A), V_(initial) (FIG. 71B),V_(stationary) (FIG. 71C) and clot size after 60 minutes (FIG. 71D) inhemophilia A plasma activated with low surface tissue factor density(n=3). An asterisk indicates a statistically significantdifference±ARC19499 (P<0.05).

FIG. 72 is a series of graphs showing the lag time (FIG. 72A),V_(initial) (FIG. 72B), V_(stationary) (FIG. 72C) and clot size after 60minutes (FIG. 72D) in hemophilia A plasma from Patient 4 activated withmedium surface tissue factor density. Each parameter is plotted as afunction of ARC19499 (squares) or recombinant factor VIIa (rVIIa;circles).

FIG. 73 is a series of graphs showing the lag time (FIG. 73A),V_(initial) (FIG. 73B), V_(stationary) (FIG. 73C) and clot size after 60minutes (FIG. 73D) in hemophilia A plasma from Patient 5 activated withmedium surface tissue factor density. Each parameter is plotted as afunction of ARC19499 (squares) or recombinant factor VIIa (rVIIa;circles).

FIG. 74 is a series of graphs showing the lag time (FIG. 74A),V_(initial) (FIG. 74B), V_(stationary) (FIG. 74C) and clot size after 60minutes (FIG. 74D) in hemophilia A plasma from Patient 6 activated withmedium surface tissue factor density. Each parameter is plotted as afunction of ARC19499 (squares) or recombinant factor VIIa (rVIIa;circles).

FIG. 75 is a series of graphs illustrating the effect of 300 nM ARC19499on the average lag time (FIG. 75A), V_(initial) (FIG. 75B),V_(stationary) (FIG. 75C) and clot size after 60 minutes (FIG. 75D) inhemophilia A plasma activated with medium surface tissue factor density(n=3).

FIG. 76 is a series of graphs illustrating the lag time (FIG. 76A),V_(initial) (FIG. 76B), V_(stationary) (FIG. 76C) and clot size after 60minutes (FIG. 76D) in normal plasma compared to hemophilia A plasma orhemophilia A plasma containing 300 nM ARC19499, activated with lowsurface tissue factor density.

FIG. 77 is a bar graph illustrating the efficiency of ARC19499 inpromoting clot propagation in normal plasma (solid bars) versushemophilia A plasma (hatched bars) as reflected in the lag time (white),V_(initial) (light grey), V_(stationary) (medium grey) and clot sizeafter 60 minutes (black). Efficiency is defined as the ratio of theparameter determined in the presence of 300 nM ARC19499 to the parameterin the absence of ARC19499.

FIG. 78 illustrates the concentration dependence of the lag time (FIG.78A) and clot size at 60 minutes (FIG. 78B) on ARC19499 in hemophilia Aplasma activated with low surface tissue factor density. These data wereused to calculate the IC₅₀ values shown in the table below the graphs.

FIG. 79 is a series of graphs illustrating the lag time (FIG. 79A),V_(initial) (FIG. 79B), V_(stationary) (FIG. 79C) and clot size after 60minutes (FIG. 79D) in hemophilia A plasma alone compared to hemophilia Aplasma containing 300 nM ARC19499 or 300 nM recombinant factor VIIa(rVIIa), activated with low surface tissue factor density.

FIG. 80 compares the lag time (FIG. 80A) and V_(initial) (FIG. 80B) inTFPI depleted plasma activated with low surface tissue factor density.Each graph shows parameters measured in TFPI depleted plasma alone(“PBS”), TFPI depleted plasma supplemented with ±10 nM recombinant TFPI(“TFPI”), TFPI depleted plasma containing 300 nM ARC19499 (“ARC”), andTFPI depleted plasma supplemented with 10 nM recombinant TFPI and 300 nMARC19499 (“ARC+TFPI”).

FIG. 81 is a series of tables showing the effects of ARC19499 on theTF-activated clotting time (TF-ACT) assay in whole blood samples fromnormal, severe hemophilia B and severe hemophilia A individuals.

FIG. 82 is a series of tables showing the effects of ARC19499 on thedilute prothrombin time (dPT) assay in plasma samples from normal,severe hemophilia B and severe hemophilia A individuals.

FIG. 83 shows the effect of different ARC19499 concentrations on ROTEMparameters in whole blood samples (without corn trypsin inhibitor (CTI))from hemophilia patients (filled squares) and healthy controls (emptycircles). The following parameters were analyzed: the clotting time(CT), the clot formation time (CFT), the maximum clot firmness (MCF) andthe alpha angle (alpha).

FIG. 84 shows the effect of different ARC19499 concentrations on theclotting time (CT) in blood samples from healthy controls (emptycircles) compared to hemophilia A patients stratified according to FVIIIlevel: <1% (filled squares), 1-5% (filled, inverted triangles), >5%(filled triangles). The hatched region indicates the range in healthycontrols.

FIG. 85 shows the effect of different ARC19499 concentrations on ROTEMparameters in whole blood samples (with corn trypsin inhibitor (CTI))from hemophilia patients (filled squares) and healthy controls (emptycircles). The following parameters were analyzed: the clotting time(CT), the clot formation time (CFT), the maximum clot firmness (MCF) andthe alpha angle (alpha).

FIG. 86 shows the effect of different ARC19499 concentrations on ROTEMparameters in whole blood samples from a single patient with acquiredhemophilia A. The following parameters were analyzed: the clotting time(CT), the clot formation time (CFT), the maximum clot firmness (MCF) andthe alpha angle (alpha).

FIG. 87 shows ROTEM parameters for healthy control blood pre-incubatedwith a neutralizing FVIII antibody. Graphs show clotting time (CT) (leftpanel) and clot formation time (CFT) (right panel) in the same controls;on the left side of each graph, values after inhibition by an FVIIIantibody are depicted.

FIG. 88 shows thrombin generation curves from the calibrated automatedthrombogram (CAT) assay in plasma from a representative severehemophilia A patient (left panel) and a healthy control (right panel).Both graphs show results in the presence (empty circles) and absence(filled squares) of 200 nM ARC19499.

FIG. 89 shows plots of calibrated automated thrombogram (CAT) parametersversus ARC19499 concentration, including the endogenous thrombinpotential (ETP), time to peak, peak thrombin concentration and starttail. In each graph, the response to ARC19499 in plasma from hemophiliapatients (filled squares) is compared to healthy controls (emptycircles).

FIG. 90 is a plot of calibrated automated thrombogram (CAT) lag timeversus ARC19499 concentration, comparing the response in hemophiliapatients (filled squares) to healthy controls (empty circles).

FIG. 91 shows the effect of different ARC19499 concentrations on peakthrombin in plasma samples from healthy controls (empty circles)compared to hemophilia A patients stratified according to FVIII level:<1% (filled squares), 1-5% (filled, inverted triangles), >5% (filledtriangles). The hatched region indicates the range observed in healthycontrols.

FIG. 92 shows thrombin generation curves in plasma from a single patientwith acquired hemophilia A containing 0 nM (filled squares), 2 nM(asterisks), 20 nM (empty circles) or 200 nM (filled stars) of ARC19499.

FIG. 93 shows calibrated automated thrombogram (CAT) parameters forhealthy control plasma pre-incubated with a neutralizing FVIII antibody.Graphs show endogenous thrombin potential (ETP) (left panel) and peakthrombin (right panel) in the same controls; on the left side of eachgraph, values after inhibition by an FVIII antibody are depicted.

FIG. 94 shows representative calibrated automated thrombogram (CAT) datafrom a healthy volunteer (ARC HV 01).

FIG. 95 shows representative calibrated automated thrombogram (CAT) datafrom a patient with severe hemophilia A (ARC SHA 05).

FIG. 96 shows representative calibrated automated thrombogram (CAT) datafrom a patient with moderate hemophilia A (ARC MoHA 01).

FIG. 97 shows representative calibrated automated thrombogram (CAT) datafrom a patient with mild hemophilia A (ARC MiHA 03).

FIG. 98 is a series of graphs depicting median calibrated automatedthrombogram (CAT) parameters (endogenous thrombin potential (ETP), peakthrombin, lag time and time to peak) measured in fresh plasma samplesfrom patients with severe hemophilia A (empty diamonds), moderatehemophilia A (empty squares), mild hemophilia A (empty triangles) orsevere hemophilia B (filled triangles) compared to healthy controls(filled circles).

FIG. 99 is a series of graphs depicting median calibrated automatedthrombogram (CAT) parameters (endogenous thrombin potential (ETP), peakthrombin, lag time and time to peak) measured in frozen/thawed plasmasamples from patients with severe hemophilia A (empty diamonds),moderate hemophilia A (empty squares), mild hemophilia A (emptytriangles) or severe hemophilia B (filled triangles) compared to healthycontrols (filled circles).

FIG. 100 shows representative whole blood thromboelastography (TEG®)data from a healthy volunteer (ARC HV 01).

FIG. 101 shows representative whole blood thromboelastography (TEG®)data from a patient with severe hemophilia A (ARC SHA 02).

FIG. 102 shows representative whole blood thromboelastography (TEG®)data from a patient with moderate hemophilia A (ARC MoHA 01).

FIG. 103 shows representative whole blood thromboelastography (TEG®)data from a patient with mild hemophilia A (ARC MiHA 01).

FIG. 104 is a series of graphs depicting median thromboelastography(TEG®) parameters (R-time, K and angle) measured in whole blood samplesfrom patients with severe hemophilia A (empty diamonds), moderatehemophilia A (empty squares), mild hemophilia A (empty triangles) orsevere hemophilia B (filled triangles) compared to healthy controls(filled circles).

FIG. 105 shows representative plasma thromboelastography (TEG®) datafrom a patient with severe hemophilia A (ARC SHA 02).

FIG. 106 shows representative plasma thromboelastography (TEG®) datafrom a patient with moderate hemophilia A (ARC MoHA 01).

FIG. 107 shows representative plasma thromboelastography (TEG®) datafrom a patient with mild hemophilia A (ARC MiHA 03).

FIG. 108 is a series of graphs depicting median thromboelastography(TEG®) parameters (R-time, K and angle) measured in plasma samples frompatients with severe hemophilia A (empty diamonds), moderate hemophiliaA (empty squares), mild hemophilia A (empty triangles) or severehemophilia B (filled triangles) compared to healthy controls (filledcircles).

FIG. 109 is a series of graphs showing that ARC19499 activity can bereversed. ARC19499 (dashed line) improved thrombin generation in thecalibrated automated thrombogram (CAT) assay compared to hemophilia Aplasma alone (solid line), as measured by endogenous thrombin potential(ETP; FIG. 109A) and peak thrombin (FIG. 109B). Addition of ARC23085(filled diamonds), ARC23087 (empty triangles), ARC23088 (filled squares)and ARC23089 (filled triangles) can reverse this improvement atconcentrations>100 nM, reaching similar levels to the absence ofARC19499. In FIG. 109C, R-values from the thromboelastography (TEG®)assay showed that 500 nM ARC19499 shortens the R-value that is prolongedin hemophilia A plasma. 1 μM ARC23085 partially reversed thisimprovement with and without a 5 minute preincubation at 37° C. ARC23087reversed the improvement with the addition of a 5 minute preincubationat 37° C. ARC23088 showed little reversal at either condition. ARC23089also reversed the ARC19499 improvement with a 5 minute preincubation at37° C.

FIG. 110 is a series of thrombin generation curves from the calibratedautomated thrombogram (CAT) assay showing the activity of ARC19499 inhemophilia A plasma in the presence of 0.00 (FIG. 110A), 0.156 (FIG.110B), 0.312 (FIG. 110C), 0.625 (FIG. 110D), 1.25 (FIG. 110E), 2.50(FIG. 110F) or 5.00 IU/mL (FIG. 110G) low molecular weight heparin(LMWH).

FIG. 111 is a series of graphs showing the endogenous thrombin potential(ETP; (FIG. 111A) and peak thrombin (FIG. 111B) from calibratedautomated thrombogram (CAT) assays performed in hemophilia A plasma withincreasing concentrations of both ARC19499 and LMWH. The concentrationof LMWH is denoted on the x-axis in units of IU/mL. At therapeutic dosesof LMWH (≧1.25 IU/mL), the procoagulant activity of ARC19499 wasreversed.

FIG. 112 is a series of graphs showing the endogenous thrombin potential(ETP; (FIG. 112A) and peak thrombin (FIG. 112B) from calibratedautomated thrombogram (CAT) assays performed in hemophilia A plasma withincreasing concentrations of both ARC19499 and LMWH. The concentrationof LMWH is denoted on the x-axis in units of μM. The data in thesegraphs were analyzed by curve-fitting to generate estimates of LMWH IC₅₀in the presence of various ARC19499 concentrations. The IC₅₀ values maybe found in the table below the graphs.

FIG. 113 is a series of graphs showing the in vitro stability of severalTFPI aptamers in serum. The stability of ARC19498 (FIG. 113A), ARC19499(FIG. 113B), ARC19500 (FIG. 113C), ARC19501 (FIG. 113D), ARC19881 (FIG.113E) and ARC19882 (FIG. 113F) in human, monkey and rat serum weremeasured over the course of 72 hours.

FIG. 114 is a graph of a thromboelastography (TEG®) assay where plasmafrom cynomolgus monkeys that were treated previously with an anti-humanFVIII antibody was mixed with increasing concentrations of ARC19499 andassayed for activity. The solid line represents plasma from untreatedmonkeys and the dashed line represents plasma from antibody treatedmonkeys, both in the absence of aptamer. The data representsmean±standard error, with the shaded areas representing the standarderror of the non-aptamer samples.

FIG. 115 is a graph showing that regardless of treatment followingFactor VIII antibody injection in cynomolgus monkeys, Factor VIIIactivity decreased to <1% and remained there for the duration of thestudy (5.5 hours). Data represent mean±standard error, n=3−6.

FIG. 116 is a series of graphs showing prothrombin (PT) and activatedpartial thromboplastin (aPTT) times before and after ARC19499 treatmentin cynomolgus monkeys.

FIG. 117 is a series of graphs from thromboelastography (TEG®) analysisshowing that R-values (FIG. 117A), a measure of clot time; angles (FIG.117B), a measure of rate of clot formation; and maximum amplitudes (MA;FIG. 117C), a measure of clot strength, determined in monkeys treatedwith saline (filled triangles), NovoSeven® (x), 600 μg/kg ARC19499(empty squares), 300 μg/kg ARC19499 (empty triangles) or 100 μg/kgARC19499 (empty diamonds). The time course of the study is denoted onthe x-axis. Data represent mean±standard error, n=3−6.

FIG. 118 is a series of graphs from thromboelastography (TEG®) analysisshowing that R-values (FIG. 118A), a measure of clot time; angles (FIG.118B), a measure of rate of clot formation; and maximum amplitude (MA;FIG. 118C), a measure of clot strength, were determined in additionalmonkeys treated with NovoSeven® (x) or 300 μg/kg ARC19499 (triangles)for a longer time course than in FIG. 117. The time course of the studyis denoted on the x-axis. Data represent mean±standard error, n=5 forNovoSeven® treatment and n=6 for ARC19499 treatment.

FIG. 119 is a graph showing TFPI levels in cynomolgus monkeys followinga 20 mg/kg intravenous (IV, solid) or subcutaneous (SC, hatched) dose ofARC19499 in nM on the y-axis. The time course is denoted on the x-axis.The pattern of TFPI release was very similar for both IV and SC dosing.Data represent mean±standard error, n=3.

FIG. 120 shows the schedule for bleeding time assessment and relatedFVIII antibody and ARC19499 dosing and blood sampling in the non-humanprimate (NHP) bleeding model.

FIG. 121 is a series of graphs showing FVIII activity levels in plasmasamples from various dosing groups of cynomolgus monkeys treated withFVIII antibody and ARC19499: Group 1, monkeys whose bleeding times werecorrected with one dose of 1 mg/kg ARC19499 (FIG. 121A); Group 2,monkeys whose bleeding times were corrected with two doses of 1 mg/kgARC19499 (FIG. 121B); Group 3, monkey whose bleeding time was correctedwith three doses of 1 mg/kg ARC19499 (FIG. 121C); and Group 4, monkeywhose bleeding time was not corrected with three doses of 1 mg/kgARC19499 (FIG. 121D).

FIG. 122 shows mean group bleeding times for Group 1 monkeys in seconds(FIG. 122A) and in terms of % of baseline bleeding time (FIG. 122B).

FIG. 123 shows individual bleeding times for Group 1 monkeys in seconds(FIG. 123A) and in terms of % of baseline bleeding time (FIG. 123B).

FIG. 124 shows mean group bleeding times for Group 2 monkeys in seconds(FIG. 124A) and in terms of % of baseline bleeding time (FIG. 124B).

FIG. 125 shows individual bleeding times for Group 2 monkeys in seconds(FIG. 125A) and in terms of % of baseline bleeding time (FIG. 125B).

FIG. 126 shows bleeding times for the Group 3 monkey in seconds (FIG.126A) and in terms of % of baseline bleeding time (FIG. 126B).

FIG. 127 shows bleeding times for the Group 4 monkey in seconds (FIG.127A) and in terms of % of baseline bleeding time (FIG. 127B).

FIG. 128 is a graph of mean group whole blood thromboelastography (TEG®)R-values plotted against sampling timepoint for Group 1 monkeys. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the time of ARC19499 dosing is indicated by an asterisk (*).

FIG. 129 is a graph of individual whole blood thromboelastography (TEG®)R-values plotted against sampling timepoint for Group 1 monkeys. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the time of ARC19499 dosing is indicated by an asterisk (*).

FIG. 130 is a graph of mean group whole blood thromboelastography (TEG®)R-values plotted against sampling timepoint for Group 2 monkeys. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the times of ARC19499 dosing are indicated by asterisks (*).

FIG. 131 is a graph of individual whole blood thromboelastography (TEG®)R-values plotted against sampling timepoint for Group 2 monkeys. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the times of ARC19499 dosing are indicated by asterisks (*).

FIG. 132 is a graph of individual whole blood thromboelastography (TEG®)R-values plotted against sampling timepoint for the Group 3 monkey. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the times of ARC19499 dosing are indicated by asterisks (*).

FIG. 133 is a graph of individual whole blood thromboelastography (TEG®)R-values plotted against sampling timepoint for the Group 4 monkey. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the times of ARC19499 dosing are indicated by asterisks (*).

FIG. 134 is a graph of mean group plasma thromboelastography (TEG®)R-values plotted against sampling timepoint for Group 1 monkeys. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the time of ARC19499 dosing is indicated by an asterisk (*).

FIG. 135 is a graph of individual plasma thromboelastography (TEG®)R-values plotted against sampling timepoint for Group 1 monkeys. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the time of ARC19499 dosing is indicated by an asterisk (*).

FIG. 136 is a graph of mean group plasma thromboelastography (TEG®)R-values plotted against sampling timepoint for Group 2 monkeys. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the times of ARC19499 dosing are indicated by asterisks (*).

FIG. 137 is a graph of individual plasma thromboelastography (TEG®)R-values plotted against sampling timepoint for Group 2 monkeys. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the times of ARC19499 dosing are indicated by asterisks (*).

FIG. 138 is a graph of individual plasma thromboelastography (TEG®)R-values plotted against sampling timepoint for the Group 3 monkey. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the times of ARC19499 dosing are indicated by asterisks (*).

FIG. 139 is a graph of individual plasma thromboelastography (TEG®)R-values plotted against sampling timepoint for the Group 4 monkey. Thetime of anti-Factor VIII antibody dosing is indicated by a plus-sign (+)and the times of ARC19499 dosing are indicated by asterisks (*).

FIG. 140 depicts derivatives of ARC17480 that contain single andmultiple 2′-substitutions in the ARC17480 sequence. Differences relativeto ARC17480 are shaded.

FIG. 141 depicts derivatives of ARC17480 that contain a singlephosphorothioate substitution between each pair of residues in theARC17480 sequence. Each phosphorothioate is indicated by an “s” betweenthe pairs of residues in the sequence. Differences relative to ARC17480are shaded.

FIG. 142A depicts tolerated and non-tolerated 2′-substitutions mappedonto the putative secondary structure of ARC17480. FIG. 142B depictsactive ARC17480 derivatives with multiple 2′-deoxy to 2′-O Methyl and/or2′-fluoro substitutions at the four deoxycytidine residues of ARC17480(residues 9, 14, 16 and 25).

FIG. 143 depicts derivatives of ARC17480 that contain single or multipledeletions in the ARC17480 sequence. Differences relative to ARC17480 arehighlighted in black.

FIG. 144A depicts tolerated and non-tolerated single residue deletionsmapped onto the putative secondary structure of ARC17480. ARC17480 iscomprised of 2′-O Methyl (circles) and 2′-deoxy (squares) nucleotidesand is modified at its 3′-terminus with an inverted deoxythymidineresidue (3T). The corresponding double residue deletion is also depictedin cases where two adjacent nucleotides were identical. Tolerateddeletions are highlighted in gray and non-tolerated deletions arehighlighted in black. Tolerated and non-tolerated double deletions areindicated. FIG. 144B depicts active ARC17480 derivatives ARC33889 andARC33895. These molecules each have seven of the ARC17480 residuesdeleted, which are represented by black circles.

FIG. 145 depicts the results of a thrombin generation experiment with3′-truncated ARC19499 derivatives. ARC19499, ARC21383, ARC21385,ARC21387 and ARC21389 all increase thrombin generation in aconcentration-dependent manner in hemophilia A plasma, as measured byendogenous thrombin potential (ETP; FIG. 145A) and peak thrombin (FIG.145B).

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, the preferred methods andmaterials are now described. Other features, objects and advantages ofthe invention will be apparent from the description. In the description,the singular form also includes the plural unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. In the case of conflict, the present description will control.

The invention provides aptamers that bind to TFPI, which are describedherein as “TFPI aptamers”, and methods for using such aptamers in thetreatment of bleeding disorders and other TFPI-mediated pathologies,diseases and disorders, with or without other agents. In addition, theTFPI aptamers may be used before, during and/or after medicalprocedures, with or without other agents, in order to reduce orotherwise delay the progression of the complications or side effectsthereof.

Identification of Aptamers

The aptamers described herein are preferably identified through a methodknown in the art as Systematic Evolution of Ligands by EXponentialEnrichment, or SELEX™, which is shown generally in FIG. 4. Morespecifically, starting with a mixture containing a starting pool ofnucleic acids, the SELEX™ method includes steps of: (a) contacting themixture with a target under conditions favorable for binding; (b)partitioning unbound nucleic acids from those nucleic acids that havebound to the target; (c) amplifying the bound nucleic acids to yield aligand-enriched mixture of nucleic acids; and, optionally, (d)reiterating the steps of contacting, partitioning and amplifying throughas many cycles as desired to yield highly specific, high affinityaptamers to the target. In those instances where transcribed aptamers,such as RNA aptamers, are being selected, the amplification step of theSELEX™ method includes the steps of: (i) reverse transcribing thenucleic acids dissociated from the nucleic acid-target complexes orotherwise transmitting the sequence information into a corresponding DNAsequence; (ii) PCR amplification; and (iii) transcribing the PCRamplified nucleic acids or otherwise transmitting the sequenceinformation into a corresponding RNA sequence before restarting theprocess. The starting pool of nucleic acids can be modified orunmodified DNA, RNA or DNA/RNA hybrids, and acceptable modificationsinclude modifications at a base, sugar and/or internucleotide linkage.The composition of the starting pool is dependent upon the desiredproperties of the final aptamer. Selections can be performed withnucleic acid sequences incorporating modified nucleotides to, e.g.,stabilize the aptamers against degradation in vivo. For example,resistance to nuclease degradation can be greatly increased by theincorporation of modifying groups at the 2′-position.

In one embodiment, the invention provides aptamers including single 2′substitutions at all bases or combinations of 2′-OH, 2′-F, 2′-deoxy,2′—NH₂ and 2′-OMe modifications of the adenosine triphosphate (ATP),guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidinetriphosphate (TTP) and uridine triphosphate (UTP) nucleotides. Inanother embodiment, the invention provides aptamers includingcombinations of 2′-OH, 2′-F, 2′-deoxy, 2′-OMe, 2′-NH₂ and2′-methoxyethyl modifications of the ATP, GTP, CTP, TTP and UTPnucleotides. In a further embodiment, the invention provides aptamersincluding all or substantially all 2′-OMe modified ATP, GTP, CTP, TTPand/or UTP nucleotides.

In some embodiments, 2′-modified aptamers of the invention are createdusing modified polymerases, e.g., a modified RNA polymerase having arate of incorporation of modified nucleotides having bulky substituentsat the furanose 2′ position that is higher than that of wild-typepolymerases. In one embodiment, the modified RNA polymerase is a mutantT7 polymerase in which the tyrosine at position 639 has been changed tophenylalanine (Y639F). In another embodiment, the modified RNApolymerase is a mutant T7 polymerase in which the tyrosine at position639 has been changed to phenylalanine and the lysine at position 378 hasbeen changed to arginine (Y639F/K378R). In yet another embodiment, themodified RNA polymerase is a mutant T7 polymerase in which the tyrosineat position 639 has been changed to phenylalanine, the histidine atposition 784 has been changed to an alanine, and the lysine at position378 has been changed to arginine (Y639F/H784A/K378R), and thetranscription reaction mixture requires a spike of 2′-OH GTP fortranscription. In a further embodiment, the modified RNA polymerase is amutant T7 polymerase in which the tyrosine at position 639 has beenchanged to phenylalanine and the histidine at position 784 has beenchanged to an alanine (Y639F/H784A).

In one embodiment, the modified RNA polymerase is a mutant T7 polymerasein which the tyrosine at position 639 has been changed to leucine(Y639L). In another embodiment, the modified RNA polymerase is a mutantT7 polymerase in which the tyrosine at position 639 has been changed toleucine and the histidine at position 784 has been changed to an alanine(Y639L/H784A). In yet another embodiment, the modified RNA polymerase isa mutant T7 polymerase in which the tyrosine at position 639 has beenchanged to leucine, the histidine at position 784 has been changed toalanine, and the lysine at position 378 has been changed to arginine(Y639L/H784A/K378R).

Another suitable RNA polymerase having a rate of incorporation ofmodified nucleotides having bulky substituents at the furanose 2′position that is higher than that of wild-type polymerases is, forexample, a mutant T3 RNA polymerase. In one embodiment, the mutant T3RNA polymerase has a mutation at position 640, wherein the tyrosine atposition 640 is replaced with a phenylalanine (Y640F). In anotherembodiment, the mutant T3 RNA polymerase has mutations at positions 640and 785, wherein the tyrosine at position 640 is replaced with a leucineand the histidine at position 785 is replaced with an alanine(Y640L/H785A).

2′-modified oligonucleotides may be synthesized entirely of modifiednucleotides or with a subset of modified nucleotides. The modificationscan be the same or different. Some or all nucleotides may be modified,and those that are modified may contain the same modification. Forexample, all nucleotides containing the same base may have one type ofmodification, while nucleotides containing other bases may havedifferent types of modification. All purine nucleotides may have onetype of modification (or are unmodified), while all pyrimidinenucleotides have another, different type of modification (or areunmodified). In this way, transcripts, or pools of transcripts, aregenerated using any combination of modifications, including for example,ribonucleotides (2′-OH), deoxyribonucleotides (2′-deoxy), 2′-aminonucleotides (2′-NH₂), 2′-fluoro nucleotides (2′-F) and 2′-O-methyl(2′-OMe) nucleotides.

As used herein, a transcription mixture containing only 2′-OMe A, G, Cand U and/or T triphosphates (2′-OMe ATP, 2′-OMe UTP and/or 2′-OMe TTP,2′-OMe CTP and 2′-OMe GTP) is referred to as an MNA or mRmY mixture, andaptamers selected therefrom are referred to as MNA aptamers or mRmYaptamers and contain only 2′-O-methyl nucleotides. A transcriptionmixture containing 2′-OMe C and U and/or T, and 2′-OH A and G isreferred to as an “rRmY” mixture, and aptamers selected therefrom arereferred to as “rRmY” aptamers. A transcription mixture containing deoxyA and G, and 2′-OMe U and/or T, and C is referred to as a “dRmY”mixture, and aptamers selected therefrom are referred to as “dRmY”aptamers. A transcription mixture containing 2′-OMe A, C and U and/or T,and 2′-OH G is referred to as a “rGmH” mixture, and aptamers selectedtherefrom are referred to as “rGmH” aptamers. A transcription mixturealternately containing 2′-OMe A, C, U and/or T and G, and 2′-OMe A, Uand/or T, and C, and 2′-F G is referred to as an “alternating mixture”,and aptamers selected therefrom are referred to as “alternating mixture”aptamers. A transcription mixture containing 2′-OH A and G, and 2′-F Cand U and/or T is referred to as an “rRfY” mixture, and aptamersselected therefrom are referred to as “rRfY” aptamers. A transcriptionmixture containing 2′-OMe A and G, and 2′-F C and U and/or T is referredto as an “mRfY” mixture, and aptamers selected therefrom are referred toas “mRfY” aptamers. A transcription mixture containing 2′-OMe A, Uand/or T, and C, and 2′-F G is referred to as a “fGmH” mixture, andaptamers selected therefrom are referred to as “fGmH” aptamers. Atranscription mixture containing 2′-OMe A, U and/or T, C and G, where upto 10% of the G's are ribonucleotides is referred to as a “r/mGmH”mixture, and aptamers selected therefrom are referred to as “r/mGmH”aptamers. A transcription mixture containing 2′-OMe A, U and/or T, andC, and deoxy G is referred to as a “dGmH” mixture, and aptamers selectedtherefrom are referred to as “dGmH” aptamers. A transcription mixturecontaining deoxy A, and 2′-OMe C, G and U and/or T is referred to as a“dAmB” mixture, and aptamers selected therefrom are referred to as“dAmB” aptamers. A transcription mixture containing 2′-OH A, and 2′-OMeC, G and U and/or T is referred to as a “rAmB” mixture, and aptamersselected therefrom are referred to as “rAmB” aptamers. A transcriptionmixture containing 2′-OH A and 2′-OH G, and 2′-deoxy C and 2′-deoxy T isreferred to as an rRdY mixture, and aptamers selected therefrom arereferred to as “rRdY” aptamers. A transcription mixture containing2′-OMe A, U and/or T, and G, and deoxy C is referred to as a “dCmD”mixture, and aptamers selected there from are referred to as “dCmD”aptamers. A transcription mixture containing 2′-OMe A, G and C, anddeoxy T is referred to as a “dTmV” mixture, and aptamers selected therefrom are referred to as “dTmV” aptamers. A transcription mixturecontaining 2′-OMe A, C and G, and 2′-OH U is referred to as a “rUmV”mixture, and aptamers selected there from are referred to as “rUmV”aptamers. A transcription mixture containing 2′-OMe A, C and G, and2′-deoxy U is referred to as a “dUmV” mixture, and aptamers selectedtherefrom are referred to as “dUmV” aptamers. A transcription mixturecontaining all 2′-OH nucleotides is referred to as a “rN” mixture, andaptamers selected therefrom are referred to as “rN”, “rRrY” or RNAaptamers. A transcription mixture containing all deoxy nucleotides isreferred to as a “dN” mixture, and aptamers selected therefrom arereferred to as “dN”, “dRdY” or DNA aptamers. A transcription mixturecontaining 2′-F C and 2′-OMe A, G and U and/or T is referred to as a“fCmD” mixture, and aptamers selected therefrom are referred to as“fCmD” aptamers. A transcription mixture containing 2′-F U and 2′-OMe A,G and C is referred to as a “fUmV mixture, and aptamers selectedtherefrom are referred to as “fUmV” aptamers. A transcription mixturecontaining 2′-F A and G, and 2′-OMe C and U and/or T is referred to as a“fRmY” mixture, and aptamers selected therefrom are referred to as“fRmY” aptamers. A transcription mixture containing 2′-F A and 2′-OMe C,G and U and/or T is referred to as a “fAmB” mixture, and aptamersselected therefrom are referred to as “fAmB” aptamers.

A number of factors have been determined to be useful for optimizing thetranscription conditions used to produce the aptamers disclosed herein.For example, a leader sequence can be incorporated into the fixedsequence at the 5′ end of a DNA transcription template. The leadersequence is typically 6-15 nucleotides long, e.g., 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 nucleotides long, and may be composed of all purines,or a mixture of purine and pyrimidine nucleotides.

For compositions that contain 2′-OMe GTP, another useful factor can bethe presence or concentration of 2′-OH guanosine or guanosinemonophosphate (GMP). Transcription can be divided into two phases: thefirst phase is initiation, during which the RNA is extended by about10-12 nucleotides; the second phase is elongation, during whichtranscription proceeds beyond the addition of the first about 10-12nucleotides. It has been found that 2′-OH GMP or guanosine added to atranscription mixture containing an excess of 2′-0Me GTP is sufficientto enable the polymerase to initiate transcription. Primingtranscription with 2′-OH guanosine e.g., or GMP is useful due to thespecificity of the polymerase for the initiating nucleotide. Thepreferred concentration of GMP is 0.5 mM and even more preferably 1 mM.

Another useful factor for optimizing the incorporation of 2′-OMesubstituted nucleotides into transcripts is the use of both divalentmagnesium and manganese in the transcription mixture. Differentcombinations of concentrations of magnesium chloride and manganesechloride have been found to affect yields of 2′-O modified transcripts,the optimum concentration of the magnesium and manganese chloride beingdependent upon the concentration of NTPs in the transcription reactionmixture that complex divalent metal ions.

Other reagents that can be included in the transcription reactioninclude buffers such as N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid (HEPES) buffer, a redox reagent such as dithiothreitol (DTT), apolycation such as spermidine, spermine, a surfactant such as TritonX100, and any combinations thereof.

In one embodiment, the HEPES buffer concentration can range from 0 to 1M. The invention also contemplates the use of other buffering agentshaving a pKa between 5 and 10, including, for example,Tris-hydroxymethyl-aminomethane. In some embodiments, the DTTconcentration can range from 0 to 400 mM. The methods of the inventionalso provide for the use of other reducing agents, including, forexample, mercaptoethanol. In some embodiments, the spermidine and/orspermine concentration can range from 0 to 20 mM. In some embodiments,the PEG-8000 concentration can range from 0 to 50% (w/v). The methods ofthe invention also provide for the use of other hydrophilic polymers,including, for example, other molecular weight PEGs or otherpolyalkylene glycols. In some embodiments, the Triton X-100concentration can range from 0 to 0.1% (w/v). The methods of theinvention also provide for the use of other non-ionic detergents,including, for example, other detergents, including other Triton-Xdetergents. In some embodiments, the MgCl₂ concentration can range from0.5 mM to 50 mM. The MnCl₂ concentration can range from 0.15 mM to 15mM. In some embodiments, the 2′-OMe NTP concentration (each NTP) canrange from 5 μM to 5 mM. In some embodiments, the 2′-OH GTPconcentration can range from 0 μM to 300 μM. In some embodiments, the2′-OH GMP concentration can range from 0 to 5 mM. The pH can range frompH 6 to pH 9.

Variations of the SELEX process may also be used to identify aptamers.For example, one may use agonist SELEX, toggle SELEX, 2′-Modified SELEXor Counter SELEX. Each of these variations of the SELEX process is knownin the art.

TFPI Aptamers

The invention includes nucleic acid aptamers, preferably of 20-55nucleotides in length, that bind to tissue factor pathway inhibitor(TFPI) and which, in some embodiments, functionally modulate, e.g.,stimulate, block or otherwise inhibit or stimulate, the activity ofTFPI.

The TFPI aptamers bind at least in part to TFPI or a variant or one ormore portions (or regions) thereof. For example, the TFPI aptamers maybind to or otherwise interact with a linear portion or a conformationalportion of TFPI. A TFPI aptamer binds to or otherwise interacts with alinear portion of TFPI when the aptamer binds to or otherwise interactswith a contiguous stretch of amino acid residues that are linked bypeptide bonds. A TFPI aptamer binds to or otherwise interacts with aconformational portion of TFPI when the aptamer binds to or otherwiseinteracts with non-contiguous amino acid residues that are broughttogether by folding or other aspects of the secondary and/or tertiarystructure of the polypeptide chain.

A TFPI variant, as used herein, encompasses variants that performessentially the same function as TFPI functions, preferably includessubstantially the same structure and in some embodiments includes atleast 70% sequence identity, preferably at least 80% sequence identity,more preferably at least 90% sequence identity, and more preferably atleast 95%, 96%, 97%, 98% or 99% sequence identity to the amino acidsequence of human TFPI, which is shown in FIG. 5 as SEQ ID NO: 11.

Preferably, the TFPI aptamers bind to full length TFPI. If an aptamerbinds to one or more portions of TFPI, it is preferable that the aptamerrequire binding contacts or other interaction with a portion of TFPI, atleast in part, outside of the K1 and K2 regions, such as theK3/C-terminal region. For example, the TFPI aptamers may bind to orotherwise interact with a linear portion or a conformational portion ofTFPI. A TFPI aptamer binds to or otherwise interacts with a linearportion of TFPI when the aptamer binds to or otherwise interacts with acontiguous stretch of amino acid residues that are linked by peptidebonds. A TFPI aptamer binds to or otherwise interacts with aconformational portion of TFPI when the aptamer binds to or otherwiseinteracts with non-contiguous amino acid residues that are broughttogether by folding or other aspects of the secondary and/or tertiarystructure of the polypeptide chain. More preferably, the TFPI aptamersbind at least in part to one or more portions of mature TFPI (forexample, FIG. 3A) that are selected from the group consisting of: aminoacids 148-170, amino acids 150-170, amino acids 155-175, amino acids160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195,amino acids 180-200, amino acids 185-205, amino acids 190-210, aminoacids 195-215, amino acids 200-220, amino acids 205-225, amino acids210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245,amino acids 230-250, amino acids 235-255, amino acids 240-260, aminoacids 245-265, amino acids 250-270, amino acids 255-275, amino acids260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180,amino acids 150-185, amino acids 150-190, amino acids 150-195, aminoacids 150-200, amino acids 150-205, amino acids 150-210, amino acids150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230,amino acids 150-235, amino acids 150-240, amino acids 150-245, aminoacids 150-250, amino acids 150-255, amino acids 150-260, amino acids150-265, amino acids 150-270, amino acids 150-275, amino acids 150-276,amino acids 190-240, amino acids 190-276, amino acids 240-276, aminoacids 242-276, amino acids 161-181, amino acids 162-181, amino acids182-240, amino acids 182-241, and amino acids 182-276.

The TFPI may be from any species, but is preferably human.

The TFPI aptamers preferably comprise a dissociation constant for humanTFPI, or a variant thereof, of less than 100 μM, less than 1 μM, lessthan 500 nM, less than 100 nM, preferably 50 nM or less, preferably 25nM or less, preferably 10 nM or less, preferably 5 nM or less, morepreferably 3 nM or less, even more preferably 1 nM or less, and mostpreferably 500 pM or less. In some embodiments, the dissociationconstant is determined by dot blot titration.

The TFPI aptamers may be ribonucleic acid, deoxyribonucleic acid,modified nucleic acids (for example, 2′-modified) or mixed ribonucleicacid, deoxyribonucleic acid and modified nucleic acids, or anycombination thereof. The aptamers may be single stranded ribonucleicacid, deoxyribonucleic acid, modified nucleic acids (for example,2′-modified), ribonucleic acid and modified nucleic acid,deoxyribonucleic acid and modified nucleic acid, or mixed ribonucleicacid, deoxyribonucleic acid and modified nucleic acids, or anycombination thereof.

In some embodiments, the TFPI aptamers comprise at least one chemicalmodification. In some embodiments, the chemical modification is selectedfrom the group consisting of: a chemical substitution at a sugarposition, a chemical substitution at an internucleotide linkage and achemical substitution at a base position. In other embodiments, thechemical modification is selected from the group consisting of:incorporation of a modified nucleotide; a 3′ cap; a 5′ cap; conjugationto a high molecular weight, non-immunogenic compound; conjugation to alipophilic compound; incorporation of a CpG motif; and incorporation ofa phosphorothioate or phosphorodithioate into the phosphate backbone. Ina preferred embodiment, the non-immunogenic, high molecular weightcompound is polyalkylene glycol, and more preferably is polyethyleneglycol (PEG). In some embodiments, the polyethylene glycol ismethoxypolyethylene glycol (mPEG). In another preferred embodiment, the3′ cap is an inverted deoxythymidine cap.

The modifications described herein may affect aptamer stability, e.g.,incorporation of a capping moiety may stabilize the aptamer againstendonuclease degradation. Additionally, the modifications describedherein may affect the binding affinity of an aptamer to its target,e.g., site specific incorporation of a modified nucleotide orconjugation to a PEG may affect binding affinity. The effect of suchmodifications on binding affinity can be determined using a variety ofart-recognized techniques, such as, e.g., functional assays, such as anELISA, or binding assays in which labeled trace aptamer is incubatedwith varying target concentrations and complexes are captured onnitrocellulose and quantitated, to compare the binding affinities pre-and post-incorporation of a modification.

Preferably, the TFPI aptamers bind at least in part to TFPI or a variantor one or more portions thereof and act as an antagonist to inhibit thefunction of TFPI.

The TFPI aptamers completely or partially inhibit, reduce, block orotherwise modulate TFPI-mediated inhibition of blood coagulation. TheTFPI aptamers are considered to completely modulate, block, inhibit,reduce, antagonize, neutralize or otherwise interfere with TFPIbiological activity, such as TFPI-mediated inhibition of bloodcoagulation, when the level of TFPI-mediated inhibition in the presenceof the TFPI aptamer is decreased by at least 95%, e.g., by 96%, 97%,98%, 99% or 100% as compared to the level TFPI-mediated inhibition inthe absence of the TFPI aptamer. The TFPI aptamers are considered topartially modulate, block, inhibit, reduce, antagonize, neutralize orotherwise interfere with TFPI biological activity, such as TFPI-mediatedinhibition, when the level of TFPI-mediated inhibition in the presenceof the TFPI aptamer is decreased by less than 95%, e.g., 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%as compared to the level of TFPI activity in the absence of the TFPIaptamer.

Examples of aptamers that bind to and modulate the function of TFPI foruse as therapeutics and/or diagnostics include, but are not limited to,ARC26835, ARC17480, ARC19498, ARC19499, ARC19500, ARC19501, ARC31301,ARC18546, ARC19881 and ARC19882.

Preferably, the TFPI aptamers comprise one of the following nucleic acidsequences:

(ARC26835)mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 1), wherein “dN” is a deoxynucleotide and “mN” is a 2′-OMethyl containing nucleotide (which is also known in the art as a2′-OMe, 2′-methoxy or 2′-OCH₃ containing nucleotide); and(ARC17480)mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2), wherein “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide; and(ARC19498)NH₂-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 3), wherein “NH₂” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide; and(ARC19499)PEG40K—NH-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 4), wherein “NH” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide, “mN” is a 2′-O Methyl containing nucleotide and “PEG”is a polyethylene glycol; and(ARC19500)NH₂-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-NH₂(SEQ Id No: 5), Wherein “dN” is a deoxynucleotide, “mN” is a 2′-O Methylcontaining nucleotide and “NH₂” is from a hexylamine linkerphosphoramidite; and(ARC19501)PEG20K—NH-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-NH-PEG20K(SEQ Id No: 6), wherein “dN” is a deoxynucleotide, “mN” is a 2′-O Methylcontaining nucleotide, “NH” is from a hexylamine linker phosphoramiditeand “PEG” is a polyethylene glycol; and(ARC31301)mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 7), wherein “dN” is a deoxynucleotide and “mN” is a 2′-OMethyl containing nucleotide; and(ARC18546)mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 8), wherein “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide; and(ARC19881)NH₂-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 9), wherein “NH₂” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide; and(ARC19882)PEG40K—NH-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 10), wherein “NH” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide, “mN” is a 2′-O Methyl containing nucleotide and “PEG”is a polyethylene glycol.

The chemical name of ARC26835 is2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl.

The chemical name of ARC17480 is2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-(3′→3′)-2′-deoxythymidine.

The chemical name of ARC19498 is6-aminohexylyl-(1→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-(3′→3′)-2′-deoxythymidine.

The chemical name of ARC19499 isN-(methoxy-polyethyleneglycol)-6-aminohexylyl-(1→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-(3′→3′)-2′-deoxythymidine.

The chemical name of ARC19500 is6-aminohexylyl-(1→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-6-aminohexylyl.

The chemical name of ARC19501 isN-(methoxy-polyethyleneglycol)-6-aminohexylyl-(1→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-6-aminohexylyl-N-(methoxy-polyethyleneglycol).

The chemical name of ARC31301 is2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl.

The chemical name of ARC18546 is2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-(3′→3′)-2′-deoxythymidine.

The chemical name of ARC19881 is6-aminohexylyl-(1→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-(3′→3′)-2′-deoxythymidine.

The chemical name of ARC19882 isN-(methoxy-polyethyleneglycol)-6-aminohexylyl-(1→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-(3′→3′)-2′-deoxythymidine.

The TFPI aptamers of the invention may have any secondary structure.Preferably, the TFPI aptamers comprise a stem and a loop motif, such asin FIGS. 10A and B. The putative secondary structure of ARC19499 isdepicted in FIG. 10C, which comprises a stem and a loop motif.

Preferably, the TFPI aptamers are connected to one or more PEG moieties,with (FIG. 10B) or without (FIG. 10A) one or more linkers. The PEGmoieties may be any type of PEG moiety. For example, the PEG moiety maybe linear, branched, multiple branched, star shaped, comb shaped or adendrimer. In addition, the PEG moiety may have any molecular weight.Preferably, the PEG moiety has a molecular weight ranging from 5-100 kDain size. More preferably, the PEG moiety has a molecular weight rangingfrom 10-80 kDa in size. Even more preferably, the PEG moiety has amolecular weight ranging from 20-60 kDa in size. Yet even morepreferably, the PEG moiety has a molecular weight ranging from 30-50 kDain size. Most preferably, the PEG moiety has a molecular weight of 40kDa in size. The same or different PEG moieties may be connected to aTFPI aptamer. The same or different linkers or no linkers may be used toconnect the same or different PEG moieties to a TFPI aptamer

Alternatively, the TFPI aptamers may be connected to one or more PEGalternatives (rather than to one or more PEG moieties), with or withoutone or more linkers. Examples of PEG alternatives include, but are notlimited to, polyoxazoline (POZ), PolyPEG, hydroxyethylstarch (HES) andalbumin. The PEG alternative may be any type of PEG alternative, but itshould function the same as or similar to a PEG moiety, i.e., to reducerenal filtration and increase the half-life of the TFPI aptamer in thecirculation. The same or different PEG alternatives may be connected toa TFPI aptamer. The same or different linkers or no linkers may be usedto connect the same or different PEG alternatives to a TFPI aptamer.Alternatively, a combination of PEG moieties and PEG alternatives may beconnected to a TFPI aptamer, with or without one or more of the same ordifferent linkers.

Preferably, the TFPI aptamers are connected to a PEG moiety via a linker(FIG. 10B). However, the TFPI aptamers may be connected to a PEG moietydirectly, without the use of a linker (FIG. 10A). The linker may be anytype of molecule. Examples of linkers include, but are not limited to,amines, thiols and azides. For example, amines (RNH₂) and activatedesters (R′C(═O)OR″) or anhydrides (R′C(═O)OC(═O)R″) can be used aslinkers to yield an amide (R′(C═O)NR). Activated esters include, withoutlimitation, NHS (N-hydroxysuccinimide) and sulfo derivatives of NHS,nitrophenyl esters and other substituted aromatic derivatives.Anhydrides can by cyclic, such as succinic acid anhydride derivatives.Amines (RNH₂) and activated carbonates (R′OC(═O)OR″) can be used toyield carbamates (ROC(═O)NR). Activated carbamates include, withoutlimitation, NHS (N-hydroxysuccinimide) and sulfo derivatives of NHS,nitrophenyl carbamates. Amines (RNH₂) and isothiocyanates (R′N═C═S) canbe used as linkers to yield isothioureas (RNHC(═S)NHR′). Amines (RNH₂)and isocyanates (R′N═C═O) can be used as linkers to yield isoureas(RNH(C═O)NHR′). Amines (RNH₂) and acyl azides (R′(C═O)N₂) can be used aslinkers to yield amides (RNH(C═O)R′). Amines (RNH₂) and aldehydes orglyoxals (R′(C═O)H) can be used as linkers to yield imines (R′CH═NR) oramines via reduction (R′CH₂═NHR). Amines (RNH₂) and sulfonyl chlorides(R′SO₂Cl) can be used as linkers to yield sulfoamides (R′SO₂NHR). Amines(RNH₂) and epoxides and oxiranes can be used as linkers to giveα-hydroxyamines. Thiols (RSH) and iodoacetyls (R′(C═O)CH₂I) can be usedas linkers to yield thioethers (RSCH₂(O═C)R′). Thiols (RSH) andmaleimides or maleimide derivatives can be used as linkers to givethioethers. Thiols (RSH) and aziridines can be used as linkers to giveα-amine thioethers. Thiols (RSH) and acryloyl derivatives (R′CH═CH2) canbe used as linkers to give thioethers (R′CH₂CH₂SR). Thiols (RSH) anddisulfides (R′SSR″) can be used as linkers to give disulfides (RSSR′ orR″). Thiols (RSH) and vinylsulfones (CH₂═CHSO₂R′) can be used as linkersto yield thiol ethers (RSCH₂CH₂SO₂R′). Azides (RN₃) and alkynes (R′C═H)can be used as linkers to yield triazolines. Preferably, the linkercontains a phosphate group. Preferably, the linker is from a 5′-aminelinker phosphoramidite. In some embodiments, the 5′-amine linkerphosphoramidite comprises 2-18 consecutive CH₂ groups. In more preferredembodiments, the 5′-amine linker phosphoramidite comprises 2-12consecutive CH₂ groups. In even more preferred embodiments, the 5′-aminelinker phosphoramidite comprises 4-8 consecutive CH₂ groups. In mostpreferred embodiments, the 5′-amine linker phosphoramidite comprises 6consecutive CH₂ groups, i.e., is a 5′-hexylamine linker phosphoramidite.One or more of the same or different linkers or no linkers may be usedto connect one or more of the same or different PEG moieties or one ormore of the same or different PEG alternatives to a TFPI aptamer.

In preferred embodiments, an aptamer, or a salt thereof, comprising thefollowing structure is provided:

wherein HN

PO₃H is from a 5′-amine linker phosphoramidite, and the aptamer is aTFPI aptamer of the invention. The 20KPEG moiety can be any PEG moietyhaving a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is amPEG moiety having a molecular weight of 20 kDa.

In a particular embodiment, the aptamer, or a salt thereof, comprisesthe following structure:

wherein HN

PO₃H is from a 5′-amine linker phosphoramidite, and

the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2), wherein “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide. In someembodiments, the 5′-amine linker phosphoramidite comprises 2-18consecutive CH₂ groups. In more preferred embodiments, the 5′-aminelinker phosphoramidite comprises 2-12 consecutive CH₂ groups. In evenmore preferred embodiments, the 5′-amine linker phosphoramiditecomprises 4-8 consecutive CH₂ groups. In most preferred embodiments, the5′-amine linker phosphoramidite comprises 6 consecutive CH₂ groups. The20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa.Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weightof 20 kDa.

In a particular embodiment, the aptamer, or a salt thereof, comprisesthe following structure:

wherein HN

PO₃H is from a 5′-amine linker phosphoramidite, and

the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 8), wherein “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide. In someembodiments, the 5′-amine linker phosphoramidite comprises 2-18consecutive CH₂ groups. In more preferred embodiments, the 5′-aminelinker phosphoramidite comprises 2-12 consecutive CH₂ groups. In evenmore preferred embodiments, the 5′-amine linker phosphoramiditecomprises 4-8 consecutive CH₂ groups. In most preferred embodiments, the5′-amine linker phosphoramidite comprises 6 consecutive CH₂ groups. The20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa.Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weightof 20 kDa.

In alternative preferred embodiments, an aptamer, or a salt thereof,comprising the following structure is provided:

wherein HN

PO₂H is from a 5′-amine linker phosphoramidite, and the aptamer is aTFPI aptamer of the invention. The 20KPEG moiety can be any PEG moietyhaving a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is amPEG moiety having a molecular weight of 20 kDa.

In a particular alternative embodiment, the aptamer, or a salt thereof,comprises the following structure:

wherein HN

PO₂H is from a 5′-amine linker phosphoramidite, and

the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 1), wherein “dN” is a deoxynucleotide and “mN” is a 2′-OMethyl containing nucleotide. In some embodiments, the 5′-amine linkerphosphoramidite comprises 2-18 consecutive CH₂ groups. In more preferredembodiments, the 5′-amine linker phosphoramidite comprises 2-12consecutive CH₂ groups. In even more preferred embodiments, the 5′-aminelinker phosphoramidite comprises 4-8 consecutive CH₂ groups. In mostpreferred embodiments, the 5′-amine linker phosphoramidite comprises 6consecutive CH₂ groups. The 20KPEG moiety can be any PEG moiety having amolecular weight of 20 kDa. Preferably, the 20KPEG moiety is a mPEGmoiety having a molecular weight of 20 kDa.

In more preferred embodiments, an aptamer, or a salt thereof, comprisingthe following structure is provided:

wherein the aptamer is a TFPI aptamer of the invention. The 20KPEGmoiety can be any PEG moiety having a molecular weight of 20 kDa.Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weightof 20 kDa.

In a particular embodiment, the aptamer, or a salt thereof, comprisesthe following structure:

wherein the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2), wherein “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide. The20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa.Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weightof 20 kDa.

In a particular embodiment, the aptamer, or a salt thereof, comprisesthe following structure:

wherein the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 8), wherein “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide. The20KPEG moiety can be any PEG moiety having a molecular weight of 20 kDa.Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weightof 20 kDa.

In alternative more preferred embodiments, an aptamer, or a saltthereof, comprising the following structure is provided:

wherein the aptamer is a TFPI aptamer of the invention. The 20KPEGmoiety can be any PEG moiety having a molecular weight of 20 kDa.Preferably, the 20KPEG moiety is a mPEG moiety having a molecular weightof 20 kDa.

In a particular alternative embodiment, the aptamer, or a salt thereof,comprises the following structure:

wherein the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 1), wherein “dN” is a deoxynucleotide and “mN” is a 2′-OMethyl containing nucleotide. The 20KPEG moiety can be any PEG moietyhaving a molecular weight of 20 kDa. Preferably, the 20KPEG moiety is amPEG moiety having a molecular weight of 20 kDa.

In most preferred embodiments, an aptamer, or a salt thereof, comprisingthe following structure is provided:

wherein “n” is about 454 ethylene oxide units (PEG=20 kDa), and theaptamer is a TFPI aptamer of the invention. “n” is about 454 ethyleneoxide units because the number of n's may vary slightly for a PEG havinga particular molecular weight. Preferably, “n” ranges from 400-500ethylene oxide units. More preferably, “n” ranges from 425-475 ethyleneoxide units. Even more preferably, “n” ranges from 440-460 ethyleneoxide units. Most preferably, “n” is 454 ethylene oxide units.

In a particular embodiment, the aptamer, or a salt thereof, comprisesthe following structure:

wherein the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2), wherein “n” is approximately 450, “3T” is an inverteddeoxythymidine, “dN” is a deoxynucleotide and “mN” is a 2′-O Methylcontaining nucleotide. This aptamer is also known as ARC19499.

In a particular embodiment, the aptamer, or a salt thereof, comprisesthe following structure:

wherein the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-mC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-mC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 8), wherein “n” is approximately 450, “3T” is an inverteddeoxythymidine, “dN” is a deoxynucleotide and “mN” is a 2′-O Methylcontaining nucleotide. This aptamer is also known as ARC19882.

In alternative most preferred embodiments, an aptamer, or a saltthereof, comprising the following structure is provided:

wherein “n” is about 454 ethylene oxide units (PEG=20 kDa), and theaptamer is a TFPI aptamer of the invention. “n” is about 454 ethyleneoxide units because the number of n's may vary slightly for a PEG havinga particular molecular weight. Preferably, “n” ranges from 400-500ethylene oxide units. More preferably, “n” ranges from 425-475 ethyleneoxide units. Even more preferably, “n” ranges from 440-460 ethyleneoxide units. Most preferably, “n” is 454 ethylene oxide units.

In a particular alternative embodiment, the aptamer, or a salt thereof,comprises the following structure:

wherein the aptamer has the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 1), wherein “n” is approximately 450, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide. Thisaptamer is also known as ARC19501.

The invention also provides aptamers that have substantially the sameability to bind to TFPI as any one of the aptamers shown in SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers havesubstantially the same structure as any one of the aptamers shown in SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, theaptamers have substantially the same ability to bind to TFPI andsubstantially the same structure as any one of the aptamers shown in SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention also providesaptamers that have substantially the same ability to bind to TFPI andsubstantially the same ability to modulate a biological function of TFPIas any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,9 or 10. The invention further provides aptamers that have substantiallythe same ability to bind to TFPI and substantially the same ability tomodulate blood coagulation as any one of the aptamers shown in SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The invention also providesaptamers that have substantially the same structure and substantiallythe same ability to modulate a biological function of TFPI as any one ofthe aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Theinvention also provides aptamers that have substantially the samestructure and substantially the same ability to modulate bloodcoagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers havesubstantially the same ability to bind to TFPI, substantially the samestructure and substantially the same ability to modulate a biologicalfunction of TFPI as any one of the aptamers shown in SEQ ID NOs: 1, 2,3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the aptamers havesubstantially the same ability to bind to TFPI, substantially the samestructure and substantially the same ability to modulate bloodcoagulation as any one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9 or 10. As used herein, substantially the same ability tobind to TFPI means that the affinity is within one or two orders ofmagnitude of the affinity of the nucleic acid sequences and/or aptamersdescribed herein. It is well within the skill of those of ordinary skillin the art to determine whether a given sequence has substantially thesame ability to bind to TFPI. In some embodiments, the aptamer thatbinds to TFPI has a nucleic acid sequence at least 70%, 80%, 90% or 95%identical to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

The ability of an aptamer to bind to TFPI may be assessed in abinding-competition assay, e.g., as described in Example 34, in whichone of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10may be selected as the competitor acting as a control aptamer. Forexample, a suitable assay may involve incubating 10 nM human TFPI(American Diagnostica, Stamford, Conn., catalog #4500PC) with traceamounts of radiolabeled control aptamer and 5000 nM, 16667 nM, 556 nM,185 nM, 61.7 nM, 20.6 nM, 6.86 nM, 2.29 nM, 0.76 nM or 0.25 nM ofunlabeled competitor aptamer. A control aptamer is included in eachexperiment. For each molecule, the percentage of radiolabeled controlaptamer bound at each competitor aptamer concentration is used foranalysis. The percentage of radiolabeled control aptamer bound isplotted as a function of aptamer concentration and fitted to theequation y=(max/(1+x/IC₅₀))+int, where y=the percentage of radiolabeledcontrol aptamer bound, x=the concentration of aptamer, max=the maximumradiolabeled control aptamer bound, and int=the y-intercept, to generatean IC₅₀ value for binding-competition. The IC₅₀ of each aptamer iscompared to the IC₅₀ of the control aptamer evaluated in the sameexperiment. An aptamer having substantially the same ability to bind mayinclude an aptamer having an IC₅₀ that is within one or two orders ofmagnitude of the IC₅₀ of the control aptamer, and/or an aptamer havingan IC₅₀ that is not more than 5-fold greater than that of the controlaptamer evaluated in the same experiment.

The ability of an aptamer to modulate a biological function and/or tomodulate blood coagulation may be assessed in a calibrated automatedthrombogram (CAT) assay, e.g., as described in Example 34, in which oneof the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 maybe selected as a control aptamer. For example, a suitable assay mayinvolve evaluation in a CAT assay in pooled hemophilia A plasma at 500nM, 167 nM, 55.6 nM, 18.5 nM, 6.17 nM and 2.08 nM aptamer concentration.A control aptamer is included in each experiment. For each molecule, theendogenous thrombin potential (ETP) and peak thrombin values at eachaptamer concentration are used for analysis. The ETP or peak thrombinvalue for hemophilia A plasma alone is subtracted from the correspondingvalue in the presence of aptamer for each molecule at eachconcentration. Then, the corrected ETP and peak values are plotted as afunction of aptamer concentration and fitted to the equationy=(max/(1+IC₅₀/x))+int, where y=ETP or peak thrombin, x=concentration ofaptamer, max=the maximum ETP or peak thrombin, and int=the y-intercept,to generate an IC₅₀ value for both the ETP and the peak thrombin. TheIC₅₀ of each aptamer is compared to the IC₅₀ of the control aptamer thatis evaluated in the same experiment. An aptamer having substantially thesame ability to modulate a biological function and/or to modulate bloodcoagulation may include an aptamer having an IC₅₀ that is within one ortwo orders of magnitude of the IC₅₀ of the control aptamer, and/or anaptamer for which one or both of the ETP and peak thrombin IC₅₀ of thatmolecule are not more than 5-fold greater than that of the controlaptamer evaluated in the same experiment.

The ability of an aptamer to modulate a biological function and/or tomodulate blood coagulation may be assessed by evaluating inhibition ofTFPI in a Factor Xa (FXa) activity assay, e.g., as described in Example34, in which one of the aptamers shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 may be selected as a control aptamer. A suitable assay mayinvolve measuring the ability of FXa to cleave a chromogenic substratein the presence and absence of TFPI, with or without the addition ofaptamer. For example, 2 nM human FXa is incubated with 8 nM human TFPI.Then, 500 μM chromogenic substrate and aptamers are added and FXacleavage of the substrate is measured by absorbance at 405 nm (A₄₀₅) asa function of time. Aptamers are tested at 500 nM, 125 nM, 31.25 nM,7.81 nM, 1.95 nM and 0.49 nM concentrations. A control aptamer isincluded in each experiment. For each aptamer concentration, the A₄₀₅ isplotted as a function of time and the linear region of each curve isfitted to the equation y=mx+b, where y=A₄₀₅, x=the aptamerconcentration, m=the rate of substrate cleavage, and b=the y-intercept,to generate a rate of FXa substrate cleavage. The rate of FXa substratecleavage in the presence of TFPI and the absence of aptamer issubtracted from the corresponding value in the presence of both TFPI andaptamer for each molecule at each concentration. Then, the correctedrates are plotted as a function of aptamer concentration and fitted tothe equation y=(V_(max)/(1+IC₅₀/x)), where y=the rate of substratecleavage, x=concentration of aptamer, and V_(max)=the maximum rate ofsubstrate cleavage, to generate an IC₅₀ and maximum (V_(max)) value. TheIC₅₀ and V_(max) values of each aptamer are compared to the IC₅₀ andV_(max) values of the control aptamer evaluated in the same experiment.An aptamer having substantially the same ability to modulate abiological function and/or to modulate blood coagulation may include anaptamer having an IC₅₀ that is within one or two orders of magnitude ofthe IC₅₀ of the control aptamer, and/or an aptamer having an IC₅₀ thatis not more than 5-fold greater than that of the control aptamerevaluated in the same experiment, and/or an aptamer having a V_(max)value not less than 80% of the V_(max) value of the control aptamerevaluated in the same experiment.

The terms “sequence identity” or “% identity”, in the context of two ormore nucleic acid or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms or by visual inspection. Forsequence comparison, typically one sequence acts as a reference sequenceto which test sequences are compared. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2: 482 (1981); by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970);by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85: 2444 (1988); by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.); or by visual inspection (see generally, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc.and Wiley-Interscience (1987)).

One example of an algorithm that is suitable for determining percentsequence identity is the algorithm used in the basic local alignmentsearch tool (hereinafter “BLAST”), see, e.g. Altschul et al., J. Mol.Biol. 215: 403-410 (1990) and Altschul et al., Nucleic Acids Res., 15:3389-3402 (1997), which is publicly available through the NationalCenter for Biotechnology Information (hereinafter “NCBI”).

Aptamers of the invention including, but not limited to, aptamersidentified by the SELEX™ method, 2′-Modified SELEX™, minimized aptamers,optimized aptamers and chemically substituted aptamers, can bemanufactured using any oligonucleotide synthesis technique that is wellknown in the art, such as solid phase oligonucleotide synthesistechniques (see, e.g., Gualtiere, F. Ed., New Trends in SyntheticMedicinal Chemistry, Ch. 9, Chemistry of Antisense Oligonucleotides, p.261-335, 2000, Wiley-VCH, New York). The manufacturing of aptamers usingsolid phase oligonucleotide synthesis techniques can also be done atcommercial scale. Solution phase methods, such as triester synthesismethods (see, e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977) andHirose et al., Tet. Lett., 28:2449 (1978)), may also be used tomanufacture aptamers of the invention, as well as recombinant means.

In addition, a variety of functional groups can be introduced during thesolid phase synthesis. The functionality can be a simple linker thatresults in a functional group, such as an amine or thiol, or may be amore complex construct, such as a biotin or a fluorescent dye.Typically, functional group linkers or more complex moieties areintroduced using a phosphoramidite, or they can be introducedpost-synthetically (i.e., after solid phase synthesis). Alternatively,by utilizing a modified solid support, a variety of functionalities canbe introduced at the 3′-end of the oligonucleotide, thereby enabling awider variety of conjugation techniques.

The invention further provides aptamers that have been identified by theSELEX™ process, which comprises the steps of (a) contacting a mixture ofnucleic acids with TFPI under conditions in which binding occurs; (b)partitioning unbound nucleic acids from those nucleic acids that havebound to TFPI; (c) amplifying the bound nucleic acids to yield aligand-enriched mixture of nucleic acids; and, optionally, (d)reiterating the steps of binding, partitioning and amplifying through asmany cycles as desired to obtain aptamer(s) that bind to TFPI.

The invention further provides methods for identifying aptamers thatbind at least in part to or otherwise interact with one or more portionsof TFPI, which comprise the steps of (a) contacting a mixture of nucleicacids with one or more portions of TFPI under conditions in whichbinding occurs; (b) partitioning unbound nucleic acids from thosenucleic acids that have bound to TFPI; (c) amplifying the bound nucleicacids to yield a ligand-enriched mixture of nucleic acids; and,optionally, (d) reiterating the steps of contacting, partitioning andamplifying through as many cycles as desired, to obtain aptamer(s) thatbind to a portion of TFPI. This method may also include intervening oradditional cycles with binding to full-length TFPI, followed bypartitioning and amplification. For example, the TFPI aptamers may bindto or otherwise interact with a linear portion or a conformationalportion of TFPI. A TFPI aptamer binds to or otherwise interacts with alinear portion of TFPI when the aptamer binds to or otherwise interactswith a contiguous stretch of amino acid residues that are linked bypeptide bonds. A TFPI aptamer binds to or otherwise interacts with aconformational portion of TFPI when the aptamer binds to or otherwiseinteracts with non-contiguous amino acid residues that are broughttogether by folding or other aspects of the secondary and/or tertiarystructure of the polypeptide chain. Preferably, the one or more portionsof mature TFPI (for example, FIG. 3A) are selected from the groupconsisting of: amino acids 148-170, amino acids 150-170, amino acids155-175, amino acids 160-180, amino acids 165-185, amino acids 170-190,amino acids 175-195, amino acids 180-200, amino acids 185-205, aminoacids 190-210, amino acids 195-215, amino acids 200-220, amino acids205-225, amino acids 210-230, amino acids 215-235, amino acids 220-240,amino acids 225-245, amino acids 230-250, amino acids 235-255, aminoacids 240-260, amino acids 245-265, amino acids 250-270, amino acids255-275, amino acids 260-276, amino acids 148-175, amino acids 150-175,amino acids 150-180, amino acids 150-185, amino acids 150-190, aminoacids 150-195, amino acids 150-200, amino acids 150-205, amino acids150-210, amino acids 150-215, amino acids 150-220, amino acids 150-225,amino acids 150-230, amino acids 150-235, amino acids 150-240, aminoacids 150-245, amino acids 150-250, amino acids 150-255, amino acids150-260, amino acids 150-265, amino acids 150-270, amino acids 150-275,amino acids 150-276, amino acids 190-240, amino acids 190-276, aminoacids 240-276, amino acids 242-276, amino acids 161-181, amino acids162-181, amino acids 182-240, amino acids 182-241, and amino acids182-276. The aptamer preferably comprises a dissociation constant forhuman TFPI or a variant or one or more portions thereof, of less than100 μM, less than 1 μM, less than 500 nM, less than 100 nM, preferably50 nM or less, preferably 25 nM or less, preferably 10 nM or less,preferably 5 nM or less, more preferably 3 nM or less, even morepreferably 1 nM or less, and most preferably 500 pM or less.

The invention also provides methods for identifying aptamers that bindat least in part to or otherwise interact with one or more portions ofTFPI, which comprise the steps of (a) contacting a mixture of nucleicacids with full-length TFPI or one or more portions of TFPI underconditions in which binding occurs; (b) partitioning unbound nucleicacids from those nucleic acids that have bound to full-length TFPI orone or more portions of TFPI; (c) specifically eluting the bound nucleicacids with full-length TFPI or a portion of TFPI, or a ligand that bindsto full-length TFPI or a portion of TFPI; (d) amplifying the boundnucleic acids to yield a ligand-enriched mixture of nucleic acids; and,optionally, (e) reiterating the steps of contacting, partitioning,eluting and amplifying through as many cycles as desired to obtainaptamer(s) that bind to one or more portions of TFPI. For example, theTFPI aptamers may bind to or otherwise interact with a linear portion ora conformational portion of TFPI. A TFPI aptamer binds to or otherwiseinteracts with a linear portion of TFPI when the aptamer binds to orotherwise interacts with a contiguous stretch of amino acid residuesthat are linked by peptide bonds. A TFPI aptamer binds to or otherwiseinteracts with a conformational portion of TFPI when the aptamer bindsto or otherwise interacts with non-contiguous amino acid residues thatare brought together by folding or other aspects of the secondary and/ortertiary structure of the polypeptide chain. Preferably, the one or moreportions of mature TFPI (for example, FIG. 3A) are selected from thegroup consisting of: amino acids 148-170, amino acids 150-170, aminoacids 155-175, amino acids 160-180, amino acids 165-185, amino acids170-190, amino acids 175-195, amino acids 180-200, amino acids 185-205,amino acids 190-210, amino acids 195-215, amino acids 200-220, aminoacids 205-225, amino acids 210-230, amino acids 215-235, amino acids220-240, amino acids 225-245, amino acids 230-250, amino acids 235-255,amino acids 240-260, amino acids 245-265, amino acids 250-270, aminoacids 255-275, amino acids 260-276, amino acids 148-175, amino acids150-175, amino acids 150-180, amino acids 150-185, amino acids 150-190,amino acids 150-195, amino acids 150-200, amino acids 150-205, aminoacids 150-210, amino acids 150-215, amino acids 150-220, amino acids150-225, amino acids 150-230, amino acids 150-235, amino acids 150-240,amino acids 150-245, amino acids 150-250, amino acids 150-255, aminoacids 150-260, amino acids 150-265, amino acids 150-270, amino acids150-275, amino acids 150-276, amino acids 190-240, amino acids 190-276,amino acids 240-276, amino acids 242-276, amino acids 161-181, aminoacids 162-181, amino acids 182-240, amino acids 182-241, and amino acids182-276. The aptamer preferably comprises a dissociation constant forhuman TFPI or a variant or one or more portions thereof of less than 100μM, less than 1 μM, less than 500 nM, less than 100 nM, preferably 50 nMor less, preferably 25 nM or less, preferably 10 nM or less, preferably5 nM or less, more preferably 3 nM or less, even more preferably 1 nM orless, and most preferably 500 pM or less.

The invention further provides methods for identifying aptamers thatbind at least in part to or otherwise interact with one or more portionsof TFPI, which comprise the steps of (a) contacting a mixture of nucleicacids with full-length TFPI or one or more portions of TFPI underconditions in which binding occurs in the presence of a TFPI ligand (aligand that binds to TFPI) that blocks one or more epitopes on TFPI fromaptamer binding; (b) partitioning unbound nucleic acids from thosenucleic acids that have bound to full-length TFPI or one or moreportions of TFPI; (c) amplifying the bound nucleic acids to yield aligand-enriched mixture of nucleic acids; and, optionally, (d)reiterating the steps of contacting, partitioning and amplifying throughas many cycles as desired to obtain aptamer(s) that bind to one or moreportions of TFPI. In other embodiments of this method, inclusion of aTFPI ligand that blocks one or more portions on TFPI from aptamerbinding can occur during the contacting step, the partitioning step, orboth. For example, the TFPI aptamers may bind to or otherwise interactwith a linear portion or a conformational portion of TFPI. A TFPIaptamer binds to or otherwise interacts with a linear portion of TFPIwhen the aptamer binds to or otherwise interacts with a contiguousstretch of amino acid residues that are linked by peptide bonds. A TFPIaptamer binds to or otherwise interacts with a conformational portion ofTFPI when the aptamer binds to or otherwise interacts withnon-contiguous amino acid residues that are brought together by foldingor other aspects of the secondary and/or tertiary structure of thepolypeptide chain. Preferably, the one or more portions of mature TFPI(for example, FIG. 3A) are selected from the group consisting of: aminoacids 148-170, amino acids 150-170, amino acids 155-175, amino acids160-180, amino acids 165-185, amino acids 170-190, amino acids 175-195,amino acids 180-200, amino acids 185-205, amino acids 190-210, aminoacids 195-215, amino acids 200-220, amino acids 205-225, amino acids210-230, amino acids 215-235, amino acids 220-240, amino acids 225-245,amino acids 230-250, amino acids 235-255, amino acids 240-260, aminoacids 245-265, amino acids 250-270, amino acids 255-275, amino acids260-276, amino acids 148-175, amino acids 150-175, amino acids 150-180,amino acids 150-185, amino acids 150-190, amino acids 150-195, aminoacids 150-200, amino acids 150-205, amino acids 150-210, amino acids150-215, amino acids 150-220, amino acids 150-225, amino acids 150-230,amino acids 150-235, amino acids 150-240, amino acids 150-245, aminoacids 150-250, amino acids 150-255, amino acids 150-260, amino acids150-265, amino acids 150-270, amino acids 150-275, amino acids 150-276,amino acids 190-240, amino acids 190-276, amino acids 240-276, aminoacids 242-276, amino acids 161-181, amino acids 162-181, amino acids182-240, amino acids 182-241, and amino acids 182-276. The aptamerpreferably comprises a dissociation constant for human TFPI or a variantor one or more portions thereof of less than 100 μM, less than 1 μM,less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably25 nM or less, preferably 10 nM or less, preferably 5 nM or less, morepreferably 3 nM or less, even more preferably 1 nM or less, and mostpreferably 500 pM or less.

The invention further provides methods for identifying aptamers thatbind at least in part to or otherwise interact with one or more portionsof TFPI, which comprise the steps of (a) contacting a mixture of nucleicacids with full-length TFPI or one or more portions of TFPI underconditions in which binding occurs; (b) partitioning unbound nucleicacids from those nucleic acids that have bound to full-length TFPI orone or more portions of TFPI; (c) partitioning bound nucleic acids thathave a desired functional property from bound nucleic acids that do nothave a desired functional property; (d) amplifying the bound nucleicacids that have a desired functional property to yield a ligand-enrichedmixture of nucleic acids; and, optionally, (e) reiterating the steps ofcontacting, partitioning, partitioning and amplifying through as manycycles as desired to obtain aptamer(s) that bind to one or more portionsof TFPI. Steps (b) and (c) can occur sequentially or simultaneously. Forexample, the TFPI aptamers may bind to or otherwise interact with alinear portion or a conformational portion of TFPI. A TFPI aptamer bindsto or otherwise interacts with a linear portion of TFPI when the aptamerbinds to or otherwise interacts with a contiguous stretch of amino acidresidues that are linked by peptide bonds. A TFPI aptamer binds to orotherwise interacts with a conformational portion of TFPI when theaptamer binds to or otherwise interacts with non-contiguous amino acidresidues that are brought together by folding or other aspects of thesecondary and/or tertiary structure of the polypeptide chain.Preferably, the one or more portions of mature TFPI (for example, FIG.3A) are selected from the group consisting of: amino acids 148-170,amino acids 150-170, amino acids 155-175, amino acids 160-180, aminoacids 165-185, amino acids 170-190, amino acids 175-195, amino acids180-200, amino acids 185-205, amino acids 190-210, amino acids 195-215,amino acids 200-220, amino acids 205-225, amino acids 210-230, aminoacids 215-235, amino acids 220-240, amino acids 225-245, amino acids230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265,amino acids 250-270, amino acids 255-275, amino acids 260-276, aminoacids 148-175, amino acids 150-175, amino acids 150-180, amino acids150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200,amino acids 150-205, amino acids 150-210, amino acids 150-215, aminoacids 150-220, amino acids 150-225, amino acids 150-230, amino acids150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250,amino acids 150-255, amino acids 150-260, amino acids 150-265, aminoacids 150-270, amino acids 150-275, amino acids 150-276, amino acids190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276,amino acids 161-181, amino acids 162-181, amino acids 182-240, aminoacids 182-241, and amino acids 182-276. The aptamer preferably comprisesa dissociation constant for human TFPI or a variant or one or moreportions thereof of less than 100 μM, less than 1 μM, less than 500 nM,less than 100 nM, preferably 50 nM or less, preferably 25 nM or less,preferably 10 nM or less, preferably 5 nM or less, more preferably 3 nMor less, even more preferably 1 nM or less, and most preferably 500 pMor less.

The invention also provides an aptamer that binds to a human tissuefactor pathway inhibitor (TFPI) polypeptide having the amino acidsequence of SEQ ID NO: 11, wherein the aptamer modulates TFPI-mediatedinhibition of blood coagulation, and wherein the aptamer competes forbinding to TFPI with a reference aptamer comprising a nucleic acidsequence selected from the group consisting of: SEQ ID NO: 4 (ARC19499),SEQ ID NO: 1 (ARC26835), SEQ ID NO: 2 (ARC17480), SEQ ID NO: 3(ARC19498), SEQ ID NO: 5 (ARC19500), SEQ ID NO:6 (ARC19501), SEQ ID NO:7 (ARC31301), SEQ ID NO: 8 (ARC18546), SEQ ID NO: 9 (ARC19881) and SEQID NO: 10 (ARC19882). Preferably, the reference aptamer comprises thenucleic acid sequence of SEQ ID NO: 4 (ARC19499).

The invention further provides an aptamer that binds to a human tissuefactor pathway inhibitor (TFPI) polypeptide having the amino acidsequence of SEQ ID NO: 11, wherein the aptamer binds to a linear portionor a conformational portion of TFPI in which at least a portion of theregion recognized by the aptamer is different than the TFPI region boundby Factor VIIa, Factor Xa, or both Factor VIIa and Factor Xa.Preferably, the aptamer binds to one or more regions comprising at leasta portion of the amino acid sequence of SEQ ID NO: 11 selected from thegroup consisting of: amino acid residues 148-170, amino acid residues150-170, amino acid residues 155-175, amino acid residues 160-180, aminoacid residues 165-185, amino acid residues 170-190, amino acid residues175-195, amino acid residues 180-200, amino acid residues 185-205, aminoacid residues 190-210, amino acid residues 195-215, amino acid residues200-220, amino acid residues 205-225, amino acid residues 210-230, aminoacid residues 215-235, amino acid residues 220-240, amino acid residues225-245, amino acid residues 230-250, amino acid residues 235-255, aminoacid residues 240-260, amino acid residues 245-265, amino acid residues250-270, amino acid residues 255-275, amino acid residues 260-276, aminoacid residues 148-175, amino acid residues 150-175, amino acid residues150-180, amino acid residues 150-185, amino acid residues 150-190, aminoacid residues 150-195, amino acid residues 150-200, amino acid residues150-205, amino acid residues 150-210, amino acid residues 150-215, aminoacid residues 150-220, amino acid residues 150-225, amino acid residues150-230, amino acid residues 150-235, amino acid residues 150-240, aminoacid residues 150-245, amino acid residues 150-250, amino acid residues150-255, amino acid residues 150-260, amino acid residues 150-265, aminoacid residues 150-270, amino acid residues 150-275, amino acid residues150-276, amino acid residues 190-240, amino acid residues 190-276, aminoacid residues 240-276, amino acid residues 242-276, amino acid residues161-181, amino acid residues 162-181, amino acid residues 182-240, aminoacid residues 182-241, and amino acid residues 182-276. More preferably,the aptamer competes with a reference aptamer comprising the nucleicacid sequence of SEQ ID NO: 4 (ARC19499) for binding to TFPI.

The invention also provides an aptamer that binds to the same region ona human tissue factor pathway inhibitor (TFPI) polypeptide having theamino acid sequence of SEQ ID NO: 11 as the region bound by a TFPIaptamer comprising the nucleic acid sequence of SEQ ID NO: 4 (ARC19499).

The invention further provides an aptamer that binds to a region on ahuman tissue factor pathway inhibitor (TFPI) polypeptide comprising oneor more portions of SEQ ID NO: 11, wherein the one or more portions isselected from the group consisting of: amino acid residues 148-170,amino acid residues 150-170, amino acid residues 155-175, amino acidresidues 160-180, amino acid residues 165-185, amino acid residues170-190, amino acid residues 175-195, amino acid residues 180-200, aminoacid residues 185-205, amino acid residues 190-210, amino acid residues195-215, amino acid residues 200-220, amino acid residues 205-225, aminoacid residues 210-230, amino acid residues 215-235, amino acid residues220-240, amino acid residues 225-245, amino acid residues 230-250, aminoacid residues 235-255, amino acid residues 240-260, amino acid residues245-265, amino acid residues 250-270, amino acid residues 255-275, aminoacid residues 260-276, amino acid residues 148-175, amino acid residues150-175, amino acid residues 150-180, amino acid residues 150-185, aminoacid residues 150-190, amino acid residues 150-195, amino acid residues150-200, amino acid residues 150-205, amino acid residues 150-210, aminoacid residues 150-215, amino acid residues 150-220, amino acid residues150-225, amino acid residues 150-230, amino acid residues 150-235, aminoacid residues 150-240, amino acid residues 150-245, amino acid residues150-250, amino acid residues 150-255, amino acid residues 150-260, aminoacid residues 150-265, amino acid residues 150-270, amino acid residues150-275, amino acid residues 150-276, amino acid residues 190-240, aminoacid residues 190-276, amino acid residues 240-276, amino acid residues242-276, amino acid residues 161-181, amino acid residues 162-181, aminoacid residues 182-240, amino acid residues 182-241, and amino acidresidues 182-276.

The invention additionally provides an aptamer that binds to humantissue factor pathway inhibitor (TFPI) and exhibits one or more of thefollowing properties: a) competes for binding to TFPI with any one ofSEQ ID NOs: 1-10; b) inhibits TFPI inhibition of Factor Xa; c) increasesthrombin generation in hemophilia plasma; d) inhibits TFPI inhibition ofthe intrinsic tenase complex; e) restores normal hemostasis, as measuredby thromboelastography (TEG®) in whole blood and plasma; f) restoresnormal clotting, as indicated by shorter clot time, more rapid clotformation or more stable clot development, as measured bythromboelastography (TEG®) or rotational thromboelastometry (ROTEM) inwhole blood and plasma; or g) decreases the clot time, as measured bydilute prothrombin time (dPT), tissue factor activated clotting time(TF-ACT) or any other TFPI-sensitive clot-time measurement.

The invention also provides an aptamer that binds to human tissue factorpathway inhibitor wherein the aptamer competes for binding to TFPI witha reference aptamer selected from the group consisting of: SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

The invention further provides an aptamer that binds to tissue factorpathway inhibitor (TFPI) wherein the aptamer competes, either directlyor indirectly, for binding to TFPI with a reference antibody selectedfrom the group consisting of: AD4903.

The invention also provides an aptamer that binds to human tissue factorpathway inhibitor (TFPI) and comprises a stem and loop motif having thenucleotide sequence of SEQ ID NO: 4, wherein: a) any one or more ofnucleotides 1, 2, 3, 4, 6, 8, 11, 12, 13, 17, 20, 21, 22, 24, 28, 30 and32 may be modified from a 2′-OMe substitution to a 2′-deoxysubstitution; b) any one or more of nucleotides 5, 7, 15, 19, 23, 27, 29and 31 may be modified from a 2′-OMe uracil to either a 2′-deoxy uracilor a 2′-deoxy thymine; c) nucleotide 18 may be modified from a 2′-OMeuracil to a 2′-deoxy uracil; and/or d) any one or more of nucleotides14, 16 and 25 may be modified from a 2′-deoxy cytosine to either a2′-OMe cytosine or a 2′-fluoro cytosine.

The invention additionally provides an aptamer that binds to humantissue factor pathway inhibitor (TFPI) and comprises nucleotides 7-28 ofSEQ ID NO: 2.

The invention further provides a method for treating a bleeding disordercomprising administering any one of the above aptamers.

The invention further provides an aptamer that binds to tissue factorpathway inhibitor (TFPI), wherein the aptamer comprises a primarynucleic acid sequence selected from the group consisting of SEQ ID NOs.:4, 1, 2, 3, 5, 6, 7, 8, 9 and 10. A primary nucleic acid sequence of anaptamer refers solely to the nucleotides (adenine, guanine, cytosine,uracil, thymine), without any modifications (such as a 2′-O Methyl,2′-fluoro modification, 3T or PEG).

Aptamer Medicinal Chemistry

Once aptamers that bind to TFPI are identified, several techniques maybe optionally performed in order to further increase binding, stability,potency and/or functional characteristics of the identified aptamersequences.

Aptamers that bind to TFPI may be truncated in order to obtain theminimal aptamer sequence having the desired binding and/or functionalcharacteristics (also referred to herein as a “minimized construct” or a“minimized aptamer”). One method of accomplishing this is by usingfolding programs and sequence analysis, e.g., aligning clone sequencesresulting from a selection to look for conserved motifs and/orcovariation to inform the design of minimized constructs. Suitablefolding programs include, for example, the RNA structure program(Mathews, D. H.; Disney, M.D.; Childs, J. L.; Schroeder, S. J.; Zuker,M.; and Turner, D. H., “Incorporating chemical modification constraintsinto a dynamic programming algorithm for prediction of RNA secondarystructure,” 2004. Proceedings of the National Academy of Sciences, US,101, 7287-7292). Biochemical probing experiments can also be performedin order to determine the 5′ and 3′ boundaries of an aptamer sequence toinform the design of minimized constructs. Minimized constructs can thenbe chemically synthesized and tested for binding and functionalcharacteristics, as compared to the non-minimized sequence from whichthey were derived. Variants of an aptamer sequence containing a seriesof 5′, 3′ and/or internal deletions may also be directly chemicallysynthesized and tested for binding and/or functional characteristics, ascompared to the non-minimized aptamer sequence from which they werederived.

Additionally, doped reselections may be used to explore the sequencerequirements within a single active aptamer sequence or a singleminimized aptamer sequence. Doped reselections are performed using asynthetic, degenerate pool that has been designed based on the singlesequence of interest. The level of degeneracy usually varies 70% to 85%from the wild type sequence, i.e., the single sequence of interest. Ingeneral, sequences with neutral mutations are identified through thedoped reselection process, but in some cases sequence changes can resultin improvements in affinity. The composite sequence information fromclones identified using doped reselections can then be used to identifythe minimal binding motif and aid in optimization efforts.

Aptamer sequences and/or minimized aptamer sequences may also beoptimized post-SELEX™ using aptamer medicinal chemistry to performrandom or directed mutagenesis of the sequence to increase bindingaffinity and/or functional characteristics, or alternatively todetermine which positions in the sequence are essential for bindingactivity and/or functional characteristics.

Aptamer medicinal chemistry is an aptamer improvement technique in whichsets of variant aptamers are chemically synthesized. These sets ofvariants typically differ from the parent aptamer by the introduction ofa single substituent, and differ from each other by the location of thissubstituent. These variants are then compared to each other and to theparent. Improvements in characteristics may be profound enough that theinclusion of a single substituent may be all that is necessary toachieve a particular therapeutic criterion.

Alternatively the information gleaned from the set of single variantsmay be used to design further sets of variants in which more than onesubstituent is introduced simultaneously. In one design strategy, all ofthe single substituent variants are ranked, the top 4 are chosen and allpossible double (6), triple (4) and quadruple (1) combinations of these4 single substituent variants are synthesized and assayed. In a seconddesign strategy, the best single substituent variant is considered to bethe new parent and all possible double substituent variants that includethis highest-ranked single substituent variant are synthesized andassayed. Other strategies may be used, and these strategies may beapplied repeatedly such that the number of substituents is graduallyincreased while continuing to identify further-improved variants.

Aptamer medicinal chemistry may be used particularly as a method toexplore the local, rather than the global, introduction of substituents.Because aptamers are discovered within libraries that are generated bytranscription, any substituents that are introduced during the SELEX™process must be introduced globally. For example, if it is desired tointroduce phosphorothioate linkages between nucleotides then they canonly be introduced at every A (or every G, C, T, U, etc.) if globallysubstituted. Aptamers that require phosphorothioates at some As (or someG, C, T, U, etc.) (locally substituted) but cannot tolerate it at otherAs (or some G, C, T, U, etc.) cannot be readily discovered by thisprocess.

The kinds of substituents that can be utilized by the aptamer medicinalchemistry process are only limited by the ability to introduce them intoan oligomer synthesis scheme. The process is certainly not limited tonucleotides alone. Aptamer medicinal chemistry schemes may includesubstituents that introduce steric bulk, hydrophobicity, hydrophilicity,lipophilicity, lipophobicity, positive charge, negative charge, neutralcharge, zwitterions, polarizability, nuclease-resistance, conformationalrigidity, conformational flexibility, protein-binding characteristics,mass, etc. Aptamer medicinal chemistry schemes may includebase-modifications, sugar-modifications or phosphodiesterlinkage-modifications.

When considering the kinds of substituents that are likely to bebeneficial within the context of a therapeutic aptamer, it may bedesirable to introduce substitutions that fall into one or more of thefollowing categories:

-   -   (1) Substituents already present in the body, e.g., 2′-deoxy,        2′-ribo, 2′-O-methyl nucleotides, inosine or 5-methyl cytosine;    -   (2) Substituents already part of an approved therapeutic, e.g.,        2′-fluoro nucleotides; or    -   (3) Substituents that hydrolyze, degrade or metabolize to one of        the above two categories, e.g., methylphosphonate-linked        oligonucleotides or phosphorothioate-linked oligonucleotides.

The aptamers of the invention include aptamers developed through aptamermedicinal chemistry, as described herein.

Target binding affinity of the aptamers of the invention can be assessedthrough a series of binding reactions between the aptamer and the target(e.g., a protein) in which trace ³²P-labeled aptamer is incubated with adilution series of the target in a buffered medium and then analyzed bynitrocellulose filtration using a vacuum filtration manifold. Referredto herein as the dot blot binding assay, this method uses a three layerfiltration medium consisting (from top to bottom) of nitrocellulose,nylon filter and gel blot paper. Aptamer that is bound to the target iscaptured on the nitrocellulose filter, whereas the non-target boundaptamer is captured on the nylon filter. The gel blot paper is includedas a supporting medium for the other filters. Following filtration, thefilter layers are separated, dried and exposed on a phosphor screen andquantified using a phosphorimaging system. The quantified results can beused to generate aptamer binding curves from which dissociationconstants (K_(D)) can be calculated. In a preferred embodiment, thebuffered medium used to perform the binding reactions is 1× Dulbecco'sPBS (with Ca⁺⁺ and Mg⁺⁺) plus 0.1 mg/mL BSA.

Generally, the ability of an aptamer to modulate the functional activityof a target can be assessed using in vitro and in vivo models, whichwill vary depending on the biological function of the target. In someembodiments, the aptamers of the invention may inhibit a knownbiological function of the target. In other embodiments, the aptamers ofthe invention may stimulate a known biological function of the target.The functional activity of aptamers of the invention can be assessedusing in vitro and in vivo models designed to measure a known functionof TFPI.

Aptamer sequences and/or minimized aptamer sequences may also beoptimized using metabolic profile directed aptamer medicinal chemistryfor site-specific identification of cleavage sites and modifications tooptimize stability of the aptamer sequences and/or minimized aptamersequences.

Metabolic profile directed aptamer medicinal chemistry involvesincubating a parent aptamer with a test fluid to result in a mixture.Then, the mixture is analyzed to determine the rate of disappearance ofthe parent aptamer or the amount or percentage of aptamer remainingafter incubation, the specific aptamer metabolic profile and thespecific aptamer metabolite sequences. Knowledge of the sequences of thespecific metabolites formed allows one to identify the sites of nucleasecleavage based on the mass of the metabolite(s). After systematicallyconducting metabolic profiling and identifying specific aptamer cleavagesites, the method involves introducing chemical substitutions ormodifications at or near the cleavage sites that are designed tooptimize the stability of the aptamer sequences and/or minimized aptamersequences.

In one embodiment, an aptamer is identified and modified by a)incubating a parent aptamer with a test fluid to result in a mixture; b)analyzing the mixture to identify metabolites of the parent aptamer,thereby detecting at least one aptamer cleavage site in the parentaptamer; and c) introducing a chemical substitution at a positionproximal to the at least one aptamer cleavage site to result in amodified aptamer. This enhances the stability of the aptamer, and, inparticular, the stability of the aptamer to endonucleases andexonucleases.

In some embodiments, the test fluid is a biological matrix, particularlya biological matrix selected from the group consisting of one or moreof: serum; plasma; cerebral spinal fluid; tissue extracts, includingcytosolic fraction, S9 fraction and microsomal fraction; aqueous humour;vitreous humour and tissue homogenates. In some embodiments, thebiological matrix is derived from a species selected from the groupconsisting of one or more of: mouse, rat, monkey, pig, human, dog,guinea pig and rabbit. In some embodiments, the test fluid comprises atleast one purified enzyme, particularly at least one purified enzymeselected from the group consisting of: snake venom phosphodiesterase andDNAse I.

In some embodiments, the analyzing step includes analyzing the resultingaptamer using liquid chromatography and mass spectrometry, particularlyelectron spray ionization liquid chromatography mass spectrometry,polyacrylamide gel electrophoresis or capillary electrophoresis todetermine a position of at least one aptamer cleavage site. In someembodiments, the analyzing step includes analyzing the resulting aptamerusing a bioanalytical method selected from the group consisting of oneor more of: denaturing polyacrylamide gel electrophoresis (PAGE);capillary electrophoresis; high performance liquid chromatography (HPLC)and liquid chromatography-mass spectrometry (LC/MS), particularlyLC/MS/MS or LC/MS/MS/MS, and more particularly electrospray ionizationLC/MS (ESI-LC/MS), ESI-LC/MS/MS and ESI-LC/MS/MS/MS.

In some embodiments, the proximal position includes a position selectedfrom the group consisting of: a position immediately 5′ to the aptamercleavage site, a 5′ position at or within three nucleotides of theaptamer cleavage site, a position immediately 3′ to the aptamer cleavagesite, a 3′ position at or within three nucleotides of the aptamercleavage site, and at the cleaved internucleotide linkage.

In some embodiments, the chemical substitution is selected from thegroup consisting of: a chemical substitution at a sugar position; achemical substitution at a base position and a chemical substitution atan internucleotide linkage. More particularly, a substitution isselected from the group consisting of: a nucleotide substituted for adifferent nucleotide; a purine substitution for a pyrimidine; a 2′-aminesubstitution for any nucleotide; a 2′-deoxy dihydrouridine substitutionfor a uridine; a 2′-deoxy-5-methyl cytidine for a cytidine; a 2-aminopurine substitution for a purine; a phosphorothioate substituted for aphosphodiester; a phosphorodithioate substituted for a phosphodiester; a2′-deoxy nucleotide substituted for a 2′-OH nucleotide, a 2′-OMenucleotide or a 2′-fluoro nucleotide; a 2′-OMe nucleotide substitutedfor a 2′-OH nucleotide, a 2′-deoxy nucleotide, or a 2′-fluoronucleotide; a 2′-fluoro nucleotide substituted for a 2′-OH nucleotide, a2′-deoxy nucleotide or a 2′-OMe nucleotide; or a 2′-O-methoxyethylnucleotide substituted for a 2′-OH, 2′-fluoro or 2′-deoxy nucleotide; a2′-β-methoxyethyl nucleotide or deoxy nucleotide for a 2′-fluoronucleotide; and the addition of one or more PEG or other polymers orother PK or distribution-influencing entity.

In additional embodiments, the introducing step of these methods furtherincludes introducing more than one chemical substitution at one or morecleavage sites or at a single cleavage site or both.

In another embodiment, wherein more than one aptamer cleavage site isdetected, the introducing step of these methods further includesintroducing at least one chemical substitution at the associatedproximal position of the aptamer cleavage site determined to occur firstin time during the incubating step or at any other cleavage site(s) thatprovides the desired properties upon introduction of a chemicalsubstitution.

In other embodiments, these methods further include the step of testingthe stability of the modified aptamer in the test fluid. In someembodiments, aptamer stability is assessed by determining the percent ofmodified aptamer that remains intact in the test fluid as compared tothe percent of the parent aptamer that remains intact in the test fluid.In some embodiments, the percent of intact aptamer is assessed by abioanalytical method selected from the group consisting of one or moreof: denaturing polyacrylamide gel electrophoresis (PAGE); capillaryelectrophoresis; HPLC and LC/MS, particularly LC/MS/MS or LC/MS/MS/MS,and more particularly ESI-LC/MS, ESI-LC/MS/MS and ESI-LC/MS/MS/MS. Inother embodiments, the modified aptamer is more stable in the test fluidthan the parent aptamer, preferably at least 2 fold, more preferably atleast 5 fold and most preferably at least 10 fold more stable.

In additional embodiments, these methods further include determining adissociation constant or IC₅₀ of the modified aptamer for its target. Insome embodiments, chemical substitutions are introduced singly at eachposition or in various combinations in the aptamer, and the dissociationconstant or IC₅₀ for each resulting aptamer is determined. Chemicalsubstitutions are introduced at a position proximal to the aptamercleavage site such that a single chemical modification results in adissociation constant for the modified aptamer that is the same or lessthan that of the parent aptamer. In another embodiment of the invention,the method includes selecting a modified aptamer having a dissociationconstant or IC₅₀ for its target that is the same or less than that forthe parent aptamer.

In other embodiments, the modified aptamer binds to a target having abiological activity, and the method further includes testing thebiological activity of the target in the presence and absence ofmodified aptamer. In another embodiment, the method further includesselecting a modified aptamer that binds to a target having a biologicalactivity that is the same or better than that of the parent aptamer. Thebiological activity may be measured in any relevant assay, such as anELISA assay or a cell-based assay.

In some embodiments, the incubating, analyzing, introducing and testingsteps are repeated iteratively until the desired stability is achieved.

The aptamers of the invention may be routinely adapted for diagnosticpurposes according to any number of techniques employed by those skilledin the art. Diagnostic utilization may include both in vivo or in vitrodiagnostic applications. Diagnostic agents need only be able to allowthe user to identify the presence of a given target at a particularlocale or concentration. Simply the ability to form binding pairs withthe target may be sufficient to trigger a positive signal for diagnosticpurposes. Those skilled in the art would also be able to adapt anyaptamer by procedures known in the art to incorporate a labeling tag totrack the presence of such ligand. Such a tag could be used in a numberof diagnostic procedures.

Aptamers Having Immunostimulatory Motifs

The invention provides aptamers that bind to TFPI and modulate itsbiological function.

Recognition of bacterial DNA by the vertebrate immune system is basedupon the recognition of unmethylated CG dinucleotides in particularsequence contexts (“CpG motifs”). One receptor that recognizes such amotif is Toll-like receptor 9 (“TLR 9”), a member of a family ofToll-like receptors (˜10 members) that participate in the innate immuneresponse by recognizing distinct microbial components. TLR 9 isactivated by unmethylated oligodeoxynucleotide (“ODN”) CpG sequences ina sequence-specific manner. The recognition of CpG motifs triggersdefense mechanisms leading to innate and ultimately acquired immuneresponses. For example, activation of TLR 9 in mice induces activationof antigen presenting cells, up-regulation of MHC class I and IImolecules, and expression of important co-stimulatory molecules andcytokines including IL-12 and IL-23. This activation both directly andindirectly enhances B and T cell responses, including a robustup-regulation of the TH1 cytokine IFN-gamma. Collectively, the responseto CpG sequences leads to: protection against infectious diseases,improved immune response to vaccines, an effective response againstasthma, and improved antibody-dependent cell-mediated cytotoxicity.Thus, CpG ODNs can provide protection against infectious diseases,function as immuno-adjuvants or cancer therapeutics (monotherapy or incombination with a mAb or other therapies), and can decrease asthma andallergic response.

A variety of different classes of CpG motifs have been identified, eachresulting upon recognition in a different cascade of events, release ofcytokines and other molecules, and activation of certain cell types.See, e.g., CpG Motifs in Bacterial DNA and Their Immune Effects, AnnuRev. Immunol. 2002, 20:709-760, which is incorporated herein byreference. Additional immunostimulatory motifs are disclosed in thefollowing U.S. Patents, each of which is incorporated herein byreference: U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116; U.S. Pat.No. 6,429,199; U.S. Pat. No. 6,214,806; U.S. Pat. No. 6,653,292; U.S.Pat. No. 6,426,334; U.S. Pat. No. 6,514,948 and U.S. Pat. No. 6,498,148.Any of these CpG or other immunostimulatory motifs can be incorporatedinto an aptamer. The choice of aptamers is dependent upon the disease ordisorder to be treated. Preferred immunostimulatory motifs are asfollows (shown 5′ to 3′, left to right) wherein “r” designates a purine,“y” designates a pyrimidine, and “X” designates any nucleotide:AACGTTCGAG (SEQ ID NO: 12); AACGTT; ACGT; rCGy; rrCGyy; XCGX; XXCGXX;and X₁X₂CGY₁Y₂; wherein X₁ is G or A, X₂ is not C, Y₁ is not G and Y₂ ispreferably T.

In those instances where a CpG motif is incorporated into an aptamerthat binds to a specific target other than a target known to bind to CpGmotifs, and upon binding stimulates an immune response (a “non-CpGtarget”), the CpG is preferably located in a non-essential region of theaptamer. Non-essential regions of aptamers can be identified bysite-directed mutagenesis, deletion analyses and/or substitutionanalyses. However, any location that does not significantly interferewith the ability of the aptamer to bind to the non-CpG target may beused. In addition to being embedded within the aptamer sequence, the CpGmotif may be appended to either or both of the 5′ and 3′ ends orotherwise attached to the aptamer. Any location or means of attachmentmay be used as long as the ability of the aptamer to bind to the non-CpGtarget is not significantly interfered with.

As used herein, “stimulation of an immune response” can mean either (1)the induction of a specific response (e.g., induction of a Th1 response)or the production of certain molecules, or (2) the inhibition orsuppression of a specific response (e.g., inhibition or suppression ofthe Th2 response) or of certain molecules.

Aptamers of the invention, including aptamers having one or more CpG orother immunostimulatory sequences, can be identified or generated by avariety of strategies using, e.g., the SELEX™ process described herein.The incorporated immunostimulatory sequences can be DNA, RNA,substituted DNA or RNA, and/or a combination of substituted orunsubstituted DNA/RNA. In general, the strategies can be divided intotwo groups. For both groups of strategies, the CpG motifs are includedto: a) stimulate the immune response to counteract situations where arepressed immune response is relevant to disease development (i.e.,immune deficiency diseases such as AIDS), and b) to focus a stimulatedimmune response against a particular target or cell type (i.e., cancercells), or to bias an immune response towards a TH1 state and away fromTH2 or TH17 state (i.e., including CpG motifs in an aptamer against ananti-allergy target such as IgE to counteract an allergic condition).

In group one, the strategies are directed to identifying or generatingaptamers including both a CpG motif or other immunostimulatory sequenceas well as a binding site for a target, where the target (hereinafter“non-CpG target”) is a target other than one known to recognize CpGmotifs or other immunostimulatory sequences. In some embodiments of theinvention, the non-CpG target is a TFPI target. The first strategy ofthis group includes performing SELEX™ to obtain an aptamer to a specificnon-CpG target, preferably a target using an oligonucleotide poolwherein a CpG motif has been incorporated into each member of the poolas, or as part of, a fixed region, e.g., in some embodiments therandomized region of the pool members includes a fixed region having aCpG motif incorporated therein, and identifying an aptamer including aCpG motif. The second strategy of this group includes performing SELEX™to obtain an aptamer to a specific non-CpG target, and followingselection, appending a CpG motif to the 5′ and/or 3′ end or engineeringa CpG motif into a region, preferably a non-essential region, of theaptamer. The third strategy of this group includes performing SELEX™ toobtain an aptamer to a specific non-CpG target, wherein during synthesisof the pool the molar ratio of the various nucleotides is biased in oneor more nucleotide addition steps so that the randomized region of eachmember of the pool is enriched in CpG motifs, and identifying an aptamerincluding a CpG motif. The fourth strategy of this group includesperforming SELEX™ to obtain an aptamer to a specific non-CpG target, andidentifying an aptamer including a CpG motif. The fifth strategy of thisgroup includes performing SELEX™ to obtain an aptamer to a specificnon-CpG target, and identifying an aptamer which, upon binding,stimulates an immune response but that does not include a CpG motif.

In group two, the strategies are directed to identifying or generatingaptamers including a CpG motif and/or other sequences that are bound bythe receptors for the CpG motifs (e.g., TLR9 or the other toll-likereceptors) and upon binding stimulate an immune response. The firststrategy of this group includes performing SELEX™ to obtain an aptamerto a target known to bind to CpG motifs or other immunostimulatorysequences and upon binding stimulate an immune response using anoligonucleotide pool wherein a CpG motif has been incorporated into eachmember of the pool as, or as part of, a fixed region, e.g., in someembodiments the randomized region of the pool members include a fixedregion having a CpG motif incorporated therein, and identifying anaptamer including a CpG motif. The second strategy of this groupincludes performing SELEX™ to obtain an aptamer to a target known tobind to CpG motifs or other immunostimulatory sequences and upon bindingstimulate an immune response and then appending a CpG motif to the 5′and/or 3′ end or engineering a CpG motif into a region, preferably anon-essential region, of the aptamer. The third strategy of this groupincludes performing SELEX™ to obtain an aptamer to a target known tobind to CpG motifs or other immunostimulatory sequences and upon bindingstimulate an immune response, wherein during synthesis of the pool themolar ratio of the various nucleotides is biased in one or morenucleotide addition steps so that the randomized region of each memberof the pool is enriched in CpG motifs, and identifying an aptamerincluding a CpG motif. The fourth strategy of this group includesperforming SELEX™ to obtain an aptamer to a target known to bind to CpGmotifs or other immunostimulatory sequences and upon binding stimulatean immune response, and identifying an aptamer including a CpG motif.The fifth strategy of this group includes performing SELEX™ to obtain anaptamer to a target known to bind to CpG motifs or otherimmunostimulatory sequences, and identifying an aptamer which, uponbinding, stimulates an immune response but that does not include a CpGmotif.

Modulation of Pharmacokinetics and Biodistribution of AptamerTherapeutics

It is important to match the pharmacokinetic properties for alloligonucleotide-based therapeutics, including aptamers, to the desiredpharmaceutical application. Aptamers must be able to be distributed totarget organs and tissues, and remain in the body (unmodified) for aperiod of time consistent with the desired dosing regimen.

The invention provides materials and methods to affect thepharmacokinetics of aptamer compositions and, in particular, the abilityto tune aptamer pharmacokinetics. The tunability of (i.e., the abilityto modulate) aptamer pharmacokinetics is achieved through conjugation ofmodifying moieties (e.g., PEG polymers) to the aptamer and/or theincorporation of modified nucleotides (e.g., 2′-fluoro or 2′-O-methyl)or modified internucleotide linkages to alter the chemical compositionof the aptamer. The ability to tune aptamer pharmacokinetics is used inthe improvement of existing therapeutic applications, or alternatively,in the development of new therapeutic applications. For example, in sometherapeutic applications, e.g., in anti-neoplastic or acute caresettings where rapid drug clearance or turn-off may be desired, it isdesirable to decrease the residence times of aptamers in thecirculation. Alternatively, in other therapeutic applications, e.g.,maintenance therapies where systemic circulation of a therapeutic isdesired, it is desirable to increase the residence times of aptamers inthe circulation.

In addition, the tunability of aptamer pharmacokinetics is used tomodify the disposition, for example the absorption, distribution,metabolism and elimination (ADME) of an aptamer to fit its therapeuticobjective in a subject. Tunability of the pharmacokinetics of an aptamercan affect the manner and extent of absorption of the aptamer, thedistribution of an aptamer throughout the fluids and tissues of thebody, the successive metabolic transformations of the aptamer and itsmetabolite(s) and finally, the elimination of the aptamer and itsmetabolite(s). For example, in some therapeutic applications, it may bedesirable to alter the biodistribution of an aptamer therapeutic in aneffort to target a particular type of tissue or a specific organ (or setof organs), or to increase the propensity to enter specific cell types.In these applications, the aptamer therapeutic preferentiallydistributes into specific tissues and/or organs and accumulates thereinto cause a therapeutic effect. In other therapeutic applications, it maybe desirable to target tissues displaying a cellular marker or a symptomassociated with a given disease, cellular injury or other abnormalpathology, such that the aptamer therapeutic preferentially accumulatesin the affected tissue. For example, PEGylation of an aptamertherapeutic (e.g., PEGylation with a 20 kDa PEG polymer or other polymeror conjugation entity) is used to target inflamed tissues, such that thePEGylated aptamer therapeutic preferentially accumulates in the inflamedtissue.

To determine the pharmacokinetic profiles of aptamer therapeutics (e.g.,aptamer conjugates or aptamers having altered chemistries, such asmodified nucleotides), a variety of parameters are studied in normalsubjects, e.g., test animals or humans, or in diseased subjects, e.g.,TFPI-specific animal models, such as animal models of hypercoagulationor hypocoagulation, or humans with a coagulation deficiency. Suchparameters include, for example, the distribution or eliminationhalf-life (t_(1/2)), the plasma clearance (CL), the volume ofdistribution (Vss), the area under the concentration-time curve (AUC),the maximum observed serum or plasma concentration (C_(max)), and themean residence time (MRT) of an aptamer composition. As used herein, theterm “AUC” refers to the area under the plasma concentration curve of anaptamer therapeutic versus the time after aptamer administration. TheAUC value is used to estimate the exposure of the aptamer and also usedto determine the bioavailability of an aptamer after an extravascularroute of administration, such as, e.g., subcutaneous administration.Bioavailability is determined by taking the ratio of the AUC obtainedafter subcutaneous administration to the AUC obtained after intravenousadministration and normalizing them to the doses used after eachadministration (i.e., the percent ratio of aptamer administered aftersubcutaneous administration as compared to the same aptamer administeredby intravenous administration at the same dose or normalized dose). TheCL value is the measurement of the removal of the parent aptamertherapeutic from the systemic circulation. The volume of distribution(Vd) is a term that relates the amount of aptamer in the body at onetime to its plasma concentration. The Vd is used to determine how well adrug is removed from the plasma and distributed to tissues and/ororgans. A larger Vd implies wide distribution, extensive tissue binding,or both a wide distribution and extensive tissue binding. There arethree basic volumes of distribution: (i) the apparent or initial volumeof distribution at time zero obtained from back extrapolation of theconcentration-time curve; (ii) the volume calculated once distributionis complete, approximating to Vdss, where the area volume is dependentupon the elimination kinetics; and (iii) the volume of distributioncalculated once distribution is complete. The parameter that shouldideally be measured is the Vdss because this parameter is independent ofthe elimination kinetics. If the Vss for the aptamer is larger than theblood volume, it suggests that the aptamer is distributed outside of thesystemic circulation and is likely to be found in the tissues or organs.Pharmacodynamic parameters may also be used to assess drugcharacteristics.

To determine the distribution of aptamer therapeutics (e.g., aptamerconjugates or aptamers having altered chemistries, such as modifiednucleotides), a tissue distribution study or quantitative whole bodyautoradiography using a radiolabeled aptamer that is administered to anormal animal or a diseased target specific animal model is used. Theaccumulation of the radiolabeled-aptamer at a specific site can bequantified.

The pharmacokinetics and biodistribution of an aptamer described herein,such as a stabilized aptamer, can be modulated in a controlled manner byconjugating an aptamer to a modulating moiety, such as, but not limitedto, a small molecule, peptide, or polymer, or by incorporating modifiednucleotides into an aptamer. The conjugation of a modifying moietyand/or altering nucleotide chemical composition alters fundamentalaspects of aptamer residence time in circulation and distribution withinand to tissues and cells.

In addition to metabolism by nucleases, oligonucleotide therapeutics aresubject to elimination via renal filtration. As such, anuclease-resistant oligonucleotide administered intravenously typicallyexhibits an in vivo half-life of <30 minutes, unless filtration can beblocked. This can be accomplished by either facilitating rapiddistribution out of the blood stream into tissues or by increasing theapparent molecular weight of the oligonucleotide above the effectivesize cut-off for the glomerulus. Conjugation of small molecular weighttherapeutics to a PEG polymer (PEGylation), as described below, candramatically lengthen residence times of aptamers in the circulation,thereby decreasing dosing frequency and enhancing effectiveness againstvascular targets.

Modified nucleotides can also be used to modulate the plasma clearanceof aptamers. For example, an unconjugated aptamer that incorporates forexample, 2′-fluoro, 2′-OMe, and/or phosphorothioate stabilizingchemistries, which is typical of current generation aptamers as itexhibits a high degree of nuclease resistance in vitro and in vivo,displays rapid distribution into tissues, primarily into the liver andkidney, when compared to unmodified aptamer.

PAG-Derivatized Nucleic Acids

As described above and as shown in FIG. 11, derivatization of nucleicacids with high molecular weight non-immunogenic polymers has thepotential to alter the pharmacokinetic and pharmacodynamic properties ofnucleic acids making them more effective and/or safer therapeuticagents. Favorable changes in activity can include increased resistanceto degradation by nucleases, decreased filtration by the kidneys,decreased exposure to the immune system, and altered distribution of thetherapeutic through the body.

The aptamer compositions of the invention may be derivatized with one ormore polyalkylene glycol (“PAG”) moieties. Typical polymers used in theinvention include polyethylene glycol (“PEG”), also known aspolyethylene oxide (“PEO”) and polypropylene glycol (including polyisopropylene glycol). Additionally, random or block copolymers ofdifferent alkylene oxides can be used in many applications. In a commonform, a polyalkylene glycol, such as PEG, is a linear polymer terminatedat each end with hydroxyl groups: HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH.This polymer, alpha-, omega-dihydroxylpolyethylene glycol, can also berepresented as HO-PEG-OH, where it is understood that the -PEG- symbolrepresents the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n typically ranges from 4 to10,000.

PAG polymers suitable for therapeutic indications typically have theproperties of solubility in water and in many organic solvents, lack oftoxicity, and lack of immunogenicity. One use of PAGs is to covalentlyattach the polymer to insoluble molecules to make the resultingPAG-molecule “conjugate” soluble. For example, it has been shown thatthe water-insoluble drug paclitaxel, when coupled to PEG, becomeswater-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995). PAGconjugates are often used not only to enhance solubility and stability,but also to prolong the blood circulation half-life of molecules andlater distribution within the body.

The PAG derivatized compounds conjugated to the aptamers of theinvention are typically between 5 and 80 kDa in size, however any sizecan be used, the choice dependent on the aptamer and application. OtherPAG derivatized compounds of the invention are between 10 and 80 kDa insize. Still other PAG derivatized compounds of the invention are between10 and 60 kDa in size. In some embodiments, the PAG moieties derivatizedto compositions of the invention are PEG moieties having a molecularweight ranging from 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 100 kDa in size. In some embodiments, the PEGis linear PEG, while in other embodiments, the PEG is branched PEG. Instill other embodiments, the PEG is a 40 kDa branched PEG as depicted inFIG. 6. In some embodiments, the 40 kDa branched PEG is attached to the5′ end of the aptamer as depicted in FIG. 7.

Production of high molecular weight PEGs (>10 kDa) can be difficult,inefficient and expensive. To synthesize high molecular weightPEG-nucleic acid conjugates, higher molecular weight activated PEGs aregenerated. Methods for generating such molecules involve the formationof a linear activated PEG, or a branched activated PEG in which two ormore PEGs are attached to a central core carrying the activated group.The terminal portions of these higher molecular weight PEG molecules,i.e., the relatively non-reactive hydroxyl (—OH) moieties, can beactivated or converted to functional moieties for attachment of one ormore of the PEGs to other compounds at reactive sites on the compound.Branched activated PEGs will have more than two termini, and in caseswhere two or more termini have been activated, such activated highermolecular weight PEG molecules are herein referred to as,multi-activated PEGs. In some cases, not all termini in a branched PEGmolecule are activated. In cases where any two termini of a branched PEGmolecule are activated, such PEG molecule is referred to as abi-activated PEG. In some cases where only one terminus in a branchedPEG molecule is activated, such PEG molecule is referred to asmono-activated. In other cases, the linear PEG molecule is di-functionaland is sometimes referred to as “PEG diol”. The terminal portions of thePEG molecule are relatively non-reactive hydroxyl moieties, the —OHgroups, that can be activated or converted to functional moieties forattachment of the PEG to other compounds at reactive sites on thecompounds. Such activated PEG diols are referred to herein as homobi-activated PEGs. The molecules are generated using any of a variety ofart-recognized techniques. In addition to activating PEG using one ofthe previously described methods, one or both of the terminal alcoholfunctionalities of the PEG molecule can be modified to allow fordifferent types of conjugation to a nucleic acid. For example,converting one of the terminal alcohol functionalities to an amine or athiol allows access to urea and thiourethane conjugates. Otherfunctionalities include, e.g., maleimides and aldehydes.

In many applications, it is desirable to cap the PEG molecule on one endwith an essentially non-reactive moiety so that the PEG molecule ismono-functional (or mono-activated). In the case of proteintherapeutics, which generally display multiple reaction sites foractivated PEGs, homo bi-functional activated PEGs lead to extensivecross-linking, yielding poorly functional aggregates. To generatemono-activated PEGs, one hydroxyl moiety on the terminus of the PEG diolmolecule typically is substituted with a non-reactive methoxy endmoiety, —OCH₃. In this embodiment, the polymer can be represented byMeO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH and is commonly referred to as“mPEG”, where n typically ranges from 4 to 10,000.

The other, un-capped terminus of the PEG molecule typically is convertedto a reactive end moiety that can be activated for attachment at areactive site on a surface or a molecule, such as a protein, peptide oroligonucleotide.

In some cases, it is desirable to produce a hetero bi-functional PEGreagent, where one end of the PEG molecule has a reactive group, such asan N-hydroxysuccinimide or nitrophenyl carbonate, while the opposite endcontains a maleimide or other activating group. In these embodiments,two different functionalities, for example, amine and thiol, may beconjugated to the activated PEG reagent at different times.

Pharmaceutical Compositions

The invention also includes pharmaceutical compositions comprising anaptamer that binds to TFPI. In some embodiments, the compositionsinclude a therapeutically effective amount of a pharmacologically activeTFPI aptamer or a pharmaceutically acceptable salt thereof, alone or incombination, with one or more pharmaceutically acceptable carriers ordiluents.

The compositions may comprise one or more TFPI aptamers. For example,the compositions may comprise ARC19499. Alternatively, the compositionsmay comprise ARC19882. Alternatively, the compositions may compriseARC19499 and another TFPI aptamer. In embodiments where the compositionincludes at least two aptamers that can be the same aptamer or twodifferent aptamers, the aptamers may, optionally, be tethered orotherwise coupled together. Preferably, the compositions compriseARC19499, either alone or in combination with another TFPI aptamer.Alternatively, the compositions comprise a TFPI aptamer in combinationwith another agent. Preferably, the compositions comprise ARC19499 incombination with another agent.

As used herein, the terms “pharmaceutically acceptable salt” refers tosalt forms of the active compound that are prepared with counter ionsthat are non-toxic under the conditions of use and are compatible with astable formulation. Examples of pharmaceutically acceptable salts ofTFPI aptamers include hydrochlorides, sulfates, phosphates, acetates,fumarates, maleates and tartrates.

The terms “pharmaceutically acceptable carrier”, “pharmaceuticallyacceptable medium” or “pharmaceutically acceptable excipient”, as usedherein, means being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.Pharmaceutically acceptable carriers are well known in the art. Examplesof pharmaceutically acceptable carriers can be found, for example, inGoodman and Gillmans, The Pharmacological Basis of Therapeutics, latestedition.

The pharmaceutical compositions will generally include a therapeuticallyeffective amount of the active component(s) of the therapy, e.g., a TFPIaptamer of the invention that is dissolved or dispersed in apharmaceutically acceptable carrier or medium. Examples of preferredpharmaceutically acceptable carriers include, but are not limited to,physiological saline solution, phosphate buffered saline solution, andglucose solution. However it is contemplated that other pharmaceuticallyacceptable carriers may also be used. Examples of other pharmaceuticallyacceptable media or carriers include any and all solvents, dispersionmedia, coatings, antibacterial agents, antifungal agents, isotonic andabsorption delaying agents and the like. Pharmaceutically acceptablecarriers that may be used in the compositions include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances, such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat. The use of such media and agents forpharmaceutically active substances is well known in the art.

The pharmaceutical compositions may also contain pharmaceuticallyacceptable excipients, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts, or buffers for modifyingor maintaining pH, osmolarity, viscosity, clarity, color, sterility,stability, rate of dissolution or absorption of the formulation. Forsolid compositions, excipients include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate and the like.

The pharmaceutical compositions are prepared according to conventionalmixing, granulating or coating methods, and typically contain 0.1% to99.9%, for example, 0.1% to 75%, 0.1% to 50%, 0.1% to 25%, 0.1% to 10%,0.1 to 5%, preferably 1% to 50%, of the active component.

The formulation of pharmaceutical compositions is known to one of skillin the art. Typically, such compositions may be formulated asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injection;as tablets or other solids for oral administration; as time releasecapsules for slow release formulations; or in any other form currentlyused, including eye drops, creams, lotions, salves, inhalants and thelike. The compositions may also be formulated as suppositories, usingfor example, polyalkylene glycols as the carrier. In some embodiments,suppositories are prepared from fatty emulsions or suspensions. The useof sterile formulations, such as saline-based washes, by surgeons,physicians or health care workers to treat a particular area in theoperating field may also be particularly useful.

The compositions may be formulated as oral dosage forms, such astablets, capsules, pills, powders, granules, elixirs, tinctures,suspensions, syrups and emulsions. For instance, for oral administrationin the form of a tablet or capsule (e.g., a gelatin capsule), the activedrug component can be combined with an oral, non-toxic, pharmaceuticallyacceptable carrier, such as ethanol, glycerol, water and the like.Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, magnesium aluminumsilicate, starch paste, gelatin, methylcellulose, sodiumcarboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars, suchas glucose or beta-lactose, corn sweeteners, natural and synthetic gums,such as acacia, tragacanth or sodium alginate, polyethylene glycol,waxes and the like. Lubricants used in these dosage forms include sodiumoleate, sodium stearate, magnesium stearate, sodium benzoate, sodiumacetate, sodium chloride, silica, talcum, stearic acid, its magnesium orcalcium salt and/or polyethylene glycol and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum starches, agar, alginic acid or its sodium salt, oreffervescent mixtures and the like. Diluents, include, e.g., lactose,dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.

Pharmaceutical compositions can also be formulated in liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesiclesand multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids containing cholesterol, stearylamine orphosphatidylcholines. In some embodiments, a film of lipid components ishydrated with an aqueous solution of drug to a form lipid layerencapsulating the drug, as described in U.S. Pat. No. 5,262,564. Forexample, the aptamers described herein can be provided as a complex witha lipophilic compound or non-immunogenic, high molecular weight compoundconstructed using methods known in the art. Additionally, liposomes maybear aptamers on their surface for targeting and carrying cytotoxicagents internally to mediate cell killing. An example of nucleic-acidassociated complexes is provided in U.S. Pat. No. 6,011,020.

The compositions of the invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compositions ofthe invention may be coupled to a class of biodegradable polymers usefulin achieving controlled release of a drug, for example, polylactic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked oramphipathic block copolymers of hydrogels.

The compositions of the invention may also be used in conjunction withmedical devices.

The quantity of active ingredient and volume of composition to beadministered depends on the host animal to be treated. Precise amountsof active compound required for administration depend on the judgment ofthe practitioner and are peculiar to each individual.

A minimal volume of a composition required to disperse the activecompounds is typically utilized. Suitable regimes for administration arealso variable, but would be typified by initially administering thecompound and monitoring the results and then giving further controlleddoses at further intervals.

Administration

The compositions may be administered to a vertebrate, preferably amammal, and more preferably a human. The terms “patient” and “subject”are used interchangeably throughout the application, and these termsinclude both human and veterinary subjects.

In embodiments where the TFPI aptamers are antagonist aptamers, the TFPIaptamer compositions provided herein are administered to subjects in anamount effective to inhibit, reduce, block or otherwise modulateTFPI-mediated inhibition of blood coagulation. The TFPI aptamercompositions may completely or partially inhibit, reduce, block orotherwise modulate TFPI-mediated inhibition of blood coagulation. TheTFPI aptamers are considered to inhibit or otherwise modulate TFPIactivity when the aptamers cause an increase in thrombin generation(such as, for example, peak thrombin, endogenous thrombin potential orlag time) over hemophilic plasma that is equivalent to at least 1-2% offactor replacement.

The compositions may be administered by numerous routes ofadministration. Such routes of administration include, but are notlimited to, oral routes; topical routes, such as intranasally, vaginallyor rectally; and parenteral routes, such as intravenous, subcutaneous,intradermal, intramuscular, intraarticular and intrathecaladministration. Suitable routes of administration may also be used incombination, such as intravenous administration followed by subcutaneousadministration. The route of administration, however, is determined bythe attending physician. Preferably, the formulations are administeredintravenously. Most preferably, the formulations are administeredsubcutaneously.

Oral dosage forms may be administered as tablets, capsules, pills,powders, granules, elixirs, tinctures, suspensions, syrups or emulsions.

Topical dosage forms include creams, ointments, lotions, aerosol spraysand gels for intranasal vehicles, inhalants or transdermal patches.

Parenteral dosage forms include pre-filled syringes, and solutions andlyophilized powders that are reconstituted prior to administration.

The dosage regimen utilizing the aptamers of the invention is selectedin accordance with a variety of factors including type, species, age,weight, sex and medical condition of the patient; the severity of thecondition to be treated; the route of administration; the renal andhepatic function of the patient; and the particular aptamer or saltthereof employed. An ordinarily skilled physician or veterinarian canreadily determine and prescribe the effective amount of the drugrequired to prevent, counter or arrest the progress of the condition.

The pharmaceutical compositions may be administered using varioustreatment regimens. For example, the compositions may be administered asa maintenance therapy at a defined dose for a defined period of time,such as when a patient is not suffering from a bleeding episode.Alternatively, the compositions may be administered on demand, i.e., asneeded, such as when a patient is suffering from a bleeding episode. Ina further alternative embodiment, the compositions may be administeredas a combination of maintenance therapy and on demand therapy. In suchan embodiment, the compositions may be administered as a maintenancetherapy at a defined dose for a defined period of time until a bleedoccurs, in which case the dosage of the compositions would be increasedon an as needed basis until the bleeding stopped, at which point thedosage of the compositions would be decreased back to the priormaintenance level. In another such embodiment, the compositions may beadministered as a maintenance therapy at a defined dose for a definedperiod of time until a bleed occurs, in which case another bleedingdisorder therapy would be administered to the patient (such as FactorVIII) until the bleeding stopped, at which point the other bleedingdisorder therapy would be discontinued. During this entire time, thecompositions would continue to be administered as a maintenance therapy.In yet another such embodiment, the compositions may be administered asa maintenance therapy at a defined dose for a defined period of timeuntil a bleed occurs, in which case the dosage of the compositions wouldbe decreased and another bleeding disorder therapy would be administeredto the patient (such as Factor VIII) until the bleeding stopped, atwhich point the dosage of the compositions would be increased back tothe prior maintenance level and the other bleeding disorder therapywould be discontinued. In another such embodiment, another bleedingdisorder therapy (such as Factor VIII) may be administered as amaintenance therapy at a defined dose for a defined period of time untila bleed occurs, in which case the compositions would be administered tothe patient until the bleeding stopped, at which point therapy with thecompositions would be discontinued. During this entire time, the otherbleeding disorder therapy would continue to be administered as amaintenance therapy. In yet another such embodiment, another bleedingdisorder therapy (such as FVIII) may be administered as a maintenancetherapy at a defined dose for a defined period of time until a bleedoccurs, in which case the dosage of the other bleeding disorder therapywould be decreased and the compositions would be administered to thepatient until the bleeding stopped, at which point the dosage of theother bleeding disorder therapy would be increased back to the priormaintenance level and therapy with the compositions would bediscontinued.

Indications

The compositions are used to treat, prevent, delay the progression of orameliorate tissue factor pathway inhibitor (TFPI)-mediated pathologies,including the treatment of bleeding disorder pathologies involvingTFPI-mediated inhibition of blood coagulation. The pathologies to betreated, prevented, delayed or ameliorated are selected from the groupconsisting of: coagulation factor deficiencies, congenital or acquired,mild or moderate or severe, including hemophilia A (Factor VIIIdeficiency), hemophilia B (Factor IX deficiency) and hemophilia C(Factor XI deficiency); hemophilia A or B with inhibitors; other factordeficiencies (V, VII, X, XIII, prothrombin, fibrinogen); deficiency ofα2-plasmin inhibitor; deficiency of plasminogen activator inhibitor 1;multiple factor deficiency; functional factor abnormalities (e.g.,dysprothrombinemia); joint hemorrhage (hemarthrosis), including, but notlimited to, ankle, elbow and knee; spontaneous bleeding in otherlocations (muscle, gastrointestinal, mouth, etc.); hemorrhagic stroke;intracranial hemorrhage; lacerations and other hemorrhage associate withtrauma; acute traumatic coagulopathy; coagulopathy associated withcancer (e.g., acute promyelocytic leukemia); von Willebrand's Disease;disseminated intravascular coagulation; liver disease; menorrhagia;thrombocytopenia and hemorrhage associated with the use ofanticoagulants (e.g., vitamin K antagonists, FXa antagonists, etc.).

The compositions may also be administered prior to, during and/or aftera medical procedure. For example, the pharmaceutical compositions may beadministered in conjunction (before, during and/or after) with medicalprocedures, such as: prophylaxis and/or treatment associated withbleeding caused by dental procedures, orthopedic surgery including butnot limited to arthroplasty (e.g., hip replacement), surgical orradionuclide synovectomy (RSV), major surgery, venipuncture, transfusionand amputation.

Therapeutic Rationale

Without wishing to be bound by theory regarding mechanism of action, thefollowing therapeutic rationale is offered by way of example only.

Inhibitors of tissue factor pathway inhibitor (TFPI) would be expectedto enhance the generation of thrombin via the tissue factor/Factor VIIapathway (also known as the extrinsic pathway). In a normal individual,activation of the extrinsic pathway stimulates initiation of thethrombin generation response, resulting in a small amount of activatedthrombin. Following initiation, this pathway is rapidly deactivated bythe inhibitory action of TFPI. Subsequent propagation of the thrombingeneration response depends upon thrombin-mediated feedback activationof the intrinsic pathway, which includes Factor VIII (FVIII) and FactorIX (FIX). Propagation is necessary to generate a sufficiently largequantity of thrombin to catalyze the formation of a stable clot.Individuals with a deficiency of either Factor VIII (hemophilia A) orFactor IX (hemophilia B) have an impaired propagation response.Individuals with a severe deficiency (<1%) cannot produce thrombin viathe intrinsic pathway that is dependent on these proteins. Thiscondition results in the inability to produce sufficient thrombin tohave adequate platelet activation, fibrin generation and stable clotformation. However, these individuals have an intact extrinsic pathwayInhibition of TFPI could permit continuation of the initiation responseand enable propagation to occur via the extrinsic pathway, permittingsufficient thrombin generation to partially or completely replace thedeficient intrinsic pathway and thus reduce bleeding risk. Individualswith mild or moderate deficiencies in these factors, who are also atrisk for increased bleeding, may also benefit from the enhanced bloodcoagulation caused by TFPI inhibition. In addition, in patients withnormal intrinsic and extrinsic pathways who are bleeding for otherreasons, such as trauma, TFPI inhibition may provide a hemostaticstimulus that could control bleeding. Patients with other deficienciesof clotting factors, platelet deficiencies, and vascular defectsassociated with bleeding, might also benefit from a treatment that wouldinhibit TFPI.

Combination Therapy

One embodiment of the invention comprises a TFPI aptamer or a saltthereof or a pharmaceutical composition used in combination with one ormore other treatments for bleeding diseases or disorders, such as otherprocoagulant factors or other inhibitors of coagulation cascaderegulatory molecules. The pharmaceutical compositions may also beadministered in combination with another drug, such as: activatedprothrombin complex concentrates (APCC), Factor Eight Inhibitor BypassAgent (FEIBA®), recombinant Factor VIIa (e.g., Novoseven®), recombinantFactor VIII (Advate®, Kogenate®, Recombinate®, Helixate®, ReFacto®),plasma-derived Factor VIII (Humate P®, Hemofil M®), recombinant FactorIX (BeneFIX®), plasma-derived Factor IX (Bebulin VH®, Konyne®,Mononine®), cryoprecipitate, desmopressin acetate (DDAVP),epsilon-aminocaproic acid or tranexamic acid. Alternatively, thepharmaceutical compositions may be administered in combination withanother therapy, such as: blood or blood-product transfusion,plasmapheresis, immune tolerance induction therapy with high doses ofreplacement factor, immune tolerance therapy with immunosuppressiveagents (e.g., prednisone, rituximab) or pain therapy. In general, thecurrently available dosage forms of the known therapeutic agents and theuses of non-drug therapies for use in such combinations will besuitable.

“Combination therapy” (or “co-therapy”) includes the administration of aTFPI aptamer and at least a second agent or treatment as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents or treatments. Thebeneficial effect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agent or treatments. Administration of thesetherapeutic agents or treatments in combination typically is carried outover a defined time period (usually minutes, hours, days or weeksdepending upon the combination selected).

Combination therapy may, but generally is not, intended to encompass theadministration of two or more of these therapeutic agents or treatmentsas part of separate monotherapy regimens that incidentally andarbitrarily result in the combinations of the invention. Combinationtherapy is intended to embrace administration of the therapeutic agentsor treatments in a sequential manner. That is, wherein each therapeuticagent or treatment is administered at a different time, as well asadministration of these therapeutic agents or treatments, or at leasttwo of the therapeutic agents or treatments, in a substantiallysimultaneous manner. Substantially simultaneous administration can beaccomplished, for example, by administering to the subject a singleinjection having a fixed ratio of each therapeutic agent or in multiple,single injections for each of the therapeutic agents.

Sequential or substantially simultaneous administration of eachtherapeutic agent or treatment can be effected by any appropriate routeincluding, but not limited to, topical routes, oral routes, intravenousroutes, subcutaneous routes, intramuscular routes, and direct absorptionthrough mucous membrane tissues. The therapeutic agents or treatmentscan be administered by the same route or by different routes. Forexample, a first therapeutic agent or treatment of the combinationselected may be administered by injection while the other therapeuticagents or treatments of the combination may be administeredsubcutaneously. Alternatively, for example, all therapeutic agents ortreatments may be administered subcutaneously or all therapeutic agentsor treatments may be administered by injection. The sequence in whichthe therapeutic agents or treatments are administered is not criticalunless noted otherwise.

Combination therapy also can embrace the administration of thetherapeutic agent or treatments as described above in furthercombination with other biologically active ingredients. Where thecombination therapy comprises a non-drug treatment, the non-drugtreatment may be conducted at any suitable time so long as a beneficialeffect from the co-action of the combination of the therapeutic agentand non-drug treatment is achieved. For example, in appropriate cases,the beneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agent,perhaps by days or even weeks.

Reversal Agents

The invention further relates to agents that reverse the effects of theTFPI aptamers, referred to herein as “TFPI reversal agents”. The agentcan be any type of molecule, such as a protein, antibody, small moleculeorganic compound or an oligonucleotide.

Preferably, a TFPI reversal agent is a nucleic acid that is 10-15nucleotides in length. However, there are no limits to the length of thereversal agent.

Preferably, a TFPI reversal agent binds to a TFPI aptamer. A TFPIreversal agent may bind to a full length TFPI aptamer or a fragmentthereof. Such binding may be through ionic interactions, covalentbonding, complementary base pairing, hydrogen bonding, or any other typeof chemical bond. Preferably, such binding is via complementary basepairing. Without wishing to be bound by theory, a TFPI reversal agentacts by hybridizing to a TFPI aptamer, thereby disrupting the TFPIaptamer's secondary and tertiary structure and preventing the binding ofthe TFPI aptamer to TFPI. By preventing the binding of the TFPI aptamerto TFPI, the effect of the binding interaction, e.g., therapeuticeffect, and/or stimulation or inhibition of the molecular pathway, canbe modulated, providing a means of controlling the extent of the bindinginteraction and the associated effect.

A TFPI reversal agent may be a ribonucleic acid, deoxyribonucleic acidor mixed ribonucleic and deoxyribonucleic acid. Preferably, a TFPIreversal agent is single stranded. Preferably, a TFPI reversal agentcomprises all 2′-O Methyl residues and a 3′-inverted deoxythymidine.However, a TFPI reversal agent may contain any nucleotides, modified orunmodified, along with any other 3′ or 5′ modifications that may befound on aptamers.

Examples of TFPI reversal agents include, but are not limited to: SEQ IDNO: 15, which is ARC23085; SEQ ID NO: 16, which is ARC23087; SEQ ID NO:17, which is ARC23088; and SEQ ID NO: 18, which is ARC23089.

Preferably, the TFPI reversal agent is a nucleic acid comprising thestructure set forth below: mA-mG-mC-mC-mA-mA-mG-mU-mA-mU-mA-mU-mU-mC-mC(SEQ ID NO: 15), wherein “mN” is a 2′-O Methyl containing residue (whichis also known in the art as a 2′-OMe, 2′-methoxy or 2′-OCH₃ containingresidue).

Alternatively, the TFPI reversal agent is a nucleic acid comprising thestructure set forth below: mU-mA-mU-mA-mU-mA-mC-mG-mC-mA-mC-mC-mU-mA-mA(SEQ ID NO: 16), wherein “mN” is a 2′-O Methyl containing residue.

Alternatively, the TFPI reversal agent is a nucleic acid comprising thestructure set forth below: mC-mU-mA-mA-mC-mG-mA-mG-mC-mC (SEQ ID NO:17), wherein “mN” is a 2′-O Methyl containing residue.

Alternatively, the TFPI reversal agent is a nucleic acid comprising thestructure set forth below: mC-mA-mC-mC-mU-mA-mA-mC-mG-mA-mG-mC-mC-mA-mA(SEQ ID NO: 18), wherein “mN” is a 2′-O Methyl containing residue.

The chemical name of ARC23085 is2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-cytidylyl.

The chemical name of ARC23087 is2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl.

The chemical name of ARC23088 is2′-OMe-cytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-cytidylyl.

The chemical name of ARC23089 is2′-OMe-cytidylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-adenylyl.

The invention also includes TFPI reversal agents that have 70% identityor more to any one of SEQ ID NOs: 15, 16, 17 or 18. For example, theTFPI reversal agents may have 70, 75, 80, 85, 90, 95 or 100% identity toone of SEQ ID NOs: 15, 16, 17 or 18.

The invention also includes pharmaceutical compositions containing TFPIreversal agents that bind to TFPI aptamers. In some embodiments, thecompositions include an effective amount of a pharmacologically activeTFPI reversal agent or a pharmaceutically acceptable salt thereof, aloneor in combination, with one or more pharmaceutically acceptablecarriers. The compositions may contain one or more different TFPIreversal agents. The TFPI reversal agents are administered to subjectsin an amount effective to reverse the therapeutic effect of the TFPIaptamer. The compositions may be administered by numerous routes ofadministration, such as, for example, topically, intranasally orparenterally. The dosage regimen for a TFPI reversal agent will dependon a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration, the renal and hepatic function ofthe patient; the amount of TFPI aptamer used to treat a patient; and theparticular TFPI reversal agent or salt thereof employed. An ordinaryskilled physician or veterinarian can readily determine and prescribethe effective amount of the TFPI reversal agent required to reverse thetherapeutic effect of a TFPI aptamer.

The invention further includes agents that neutralize the hemostaticactivity of the TFPI aptamer. Such an agent may bind to TFPI and preventits inhibition by the TFPI aptamer, or the agent may inhibit downstreamcoagulation factors (e.g., FXa or thrombin) in a manner that counteractsthe hemostatic activity of the TFPI aptamer. Such an agent may include,but is not limited to, anticoagulants, such as unfractionated heparin orlow molecular weight heparin. A study by Wesselschmidt (Wesselschmidt etal., “Structural requirements for tissue factor pathway inhibitorinteractions with factor Xa and heparin”, Blood Coagul Fibrinolysis,vol. 4, pp. 661-669 (1993)) shows that heparin binds to TFPI throughinteractions with the K3 and C-terminal domains, which could interfereeither directly or indirectly with the ability of TFPI aptamers to bindto TFPI. Moreover, in the same study, heparin-binding was observed tofacilitate the FXa inhibitory activity of TFPI, which would tend tofurther counteract the hemostatic activity of TFPI aptamers. Finally,heparin is well known to inhibit thrombin, FXa and other coagulationfactors through an antithrombin-dependent mechanism. These activitiescould neutralize the ability of TFPI aptamers to stimulate thrombingeneration and clot formation. In the event that the hemostatic effectsof TFPI aptamers were to induce thrombosis, one of these agents could beadministered to arrest its progression. An ordinary skilled physician orveterinarian can readily determine and prescribe the effective amount ofthe anticoagulant or other neutralizing agent required to reverse thehemostatic effect of a TFPI aptamer.

Kits

The pharmaceutical compositions may also be packaged in a kit. The kitwill comprise the composition, along with instructions regardingadministration of the TFPI aptamer. The kit may also comprise one ormore of the following: a syringe or pre-filled syringe, an intravenousbag or bottle, a vial, the same TFPI aptamer in a different dosage formor another TFPI aptamer. For example, the kit may comprise both anintravenous formulation and a subcutaneous formulation of a TFPI aptamerof the invention. Alternatively, the kit may comprise lyophilized TFPIaptamer and an intravenous bag of physiological saline solution orphosphate buffered saline solution. The kit form is particularlyadvantageous when the separate components must be administered indifferent dosage forms (i.e., parenteral and oral) or are administeredat different dosage intervals. The kit may further comprise a TFPIreversal agent, along with instructions regarding administration of thereversal agent. The kit may contain both an intravenous formulation anda subcutaneous formulation of the TFPI reversal agent. Alternatively,the kit may contain lyophilized TFPI reversal agent and an intravenousbag of solution.

Preferably, the kits are stored at 5±3° C. The kits can also be storedat room temperature or frozen at −20° C.

Regulating TFPI

The invention also provides a method for regulating TFPI in which amolecule binds or otherwise interacts with one or more portions of TFPI,wherein at least one portion is outside of the K1 and K2 domains ofTFPI, such as the K3/C terminal region. The molecule can be any type ofmolecule, such as, for example, a small molecule organic compound, anantibody, a protein or peptide, a nucleic acid, a siRNA, an aptamer, orany combination thereof. Preferably, the molecule is a small moleculeorganic compound. More preferably, the molecule is an antibody. Mostpreferably, the molecule is an aptamer. For example, the molecule maybind to or otherwise interact with a linear portion or a conformationalportion of TFPI. A molecule binds to or otherwise interacts with alinear portion of TFPI when the molecule binds to or otherwise interactswith a contiguous stretch of amino acid residues that are linked bypeptide bonds. A molecule binds to or otherwise interacts with aconformational portion of TFPI when the molecule binds to or otherwiseinteracts with non-contiguous amino acid residues that are broughttogether by folding or other aspects of the secondary and/or tertiarystructure of the polypeptide chain. Preferably, the molecule binds atleast in part to one or more portions of mature TFPI (for example, FIG.3A) that are selected from the group consisting of: amino acids 148-170,amino acids 150-170, amino acids 155-175, amino acids 160-180, aminoacids 165-185, amino acids 170-190, amino acids 175-195, amino acids180-200, amino acids 185-205, amino acids 190-210, amino acids 195-215,amino acids 200-220, amino acids 205-225, amino acids 210-230, aminoacids 215-235, amino acids 220-240, amino acids 225-245, amino acids230-250, amino acids 235-255, amino acids 240-260, amino acids 245-265,amino acids 250-270, amino acids 255-275, amino acids 260-276, aminoacids 148-175, amino acids 150-175, amino acids 150-180, amino acids150-185, amino acids 150-190, amino acids 150-195, amino acids 150-200,amino acids 150-205, amino acids 150-210, amino acids 150-215, aminoacids 150-220, amino acids 150-225, amino acids 150-230, amino acids150-235, amino acids 150-240, amino acids 150-245, amino acids 150-250,amino acids 150-255, amino acids 150-260, amino acids 150-265, aminoacids 150-270, amino acids 150-275, amino acids 150-276, amino acids190-240, amino acids 190-276, amino acids 240-276, amino acids 242-276,amino acids 161-181, amino acids 162-181, amino acids 182-240, aminoacids 182-241, and amino acids 182-276. The molecule preferablycomprises a dissociation constant for human TFPI, or a variant thereof,of less than 100 μM, less than 1 μM, less than 500 nM, less than 100 nM,preferably 50 nM or less, preferably 25 nM or less, preferably 10 nM orless, preferably 5 nM or less, more preferably 3 nM or less, even morepreferably 1 nM or less, and most preferably 500 pM or less.

Many modifications and variations of the invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

In the examples, one or more of the following TFPI aptamers were used toconduct the various experiments. ARC26835 is the aptamer described inSEQ ID NO: 1. ARC17480 is the aptamer described in SEQ ID NO: 2.ARC19498 is the aptamer described in SEQ ID NO: 3. ARC19499 is theaptamer described in SEQ ID NO: 4. ARC19500 is the aptamer described inSEQ ID NO: 5. ARC19501 is the aptamer described in SEQ ID NO: 6.ARC26835 is the core aptamer sequence for each of ARC17480, ARC19498,ARC19499, ARC19500 and ARC19501. ARC31301 is the aptamer described inSEQ ID NO: 7. ARC18546 is the aptamer described in SEQ ID NO: 8.ARC19881 is the aptamer described in SEQ ID NO: 9. ARC19882 is theaptamer described in SEQ ID NO: 10. ARC31301 is the core aptamersequence for each of ARC18546, ARC19881 and ARC19882.

Example 1

This example demonstrates how ARC19499 was generated.

In vitro selection experiments were performed using a pool of modifiedoligonucleotide molecules, each of which contained dC, mA, mG and mUresidues, and recombinant human tissue factor pathway inhibitor (TFPI),which was obtained from American Diagnostica (catalog #4500PC, Stamford,Conn.). Iterative rounds of selection for binding to TFPI, followed byamplification, were performed to generate ARC14943, an 84 nucleotidelong precursor to ARC19499. ARC14943 was minimized from 84 nucleotidesto 32 nucleotides (ARC26835) using computational folding predictionprograms and systematic deletion. Appendage of a 3′-inverteddeoxythymidine residue to ARC26835 resulted in ARC17480, a 33 nucleotidelong molecule. Addition of a 5′-hexylamine group to ARC17480 resulted inARC19498. This molecule was then PEGylated via the 5′-hexylamine groupwith a 40 kDa PEG moiety to result in ARC19499.

Example 2

This example demonstrates that ARC17480 binds tightly to TFPI in vitroin a dot-blot binding experiment, in both the absence and presence ofcompetitor tRNA.

Radiolabeled ARC17480 was incubated with different concentrations ofTFPI. ARC17480 bound to TFPI was then captured on a nitrocellulosefilter membrane. The ratio of radiolabeled ARC17480 bound to thenitrocellulose filter over total radiolabeled ARC17480 added wasdetermined and plotted as the percentage of ARC17480 bound as a functionof protein concentration. An example of an ARC17480/TFPI binding plot isshown in FIG. 12A. The data were fit to models for monophasic andbiphasic aptamer-protein binding. This experiment was repeated eleventimes and K_(D)s using both monophasic and biphasic binding models weredetermined for each data set. The mean K_(D) determined using amonophasic fit was 4.0±1.5 nM and using a biphasic fit was 1.7±0.7 nM.Both monophasic and biphasic fits to the data assume different modelsfor the interaction of ARC17480 to TFPI, although the fits in and ofthemselves do not explicitly support either binding model. Regardless ofthe model used to fit the data, the K_(D) determined for binding ofARC17480 to TFPI was essentially the same. When the K_(D)s determinedfrom both the monophasic and biphasic fits were taken intoconsideration, the mean K_(D) of ARC17480 binding to TFPI was 2.9±1.6nM. This mean K_(D) does not assume a mode of binding interactionbetween ARC17480 and TFPI and, as such, is the most robust determinationof the binding interaction between the aptamer and the protein. ARC17480maintained binding to human TFPI in the presence of tRNA, indicatingthat the binding was specific. A shift in binding affinity of ARC17480to TFPI was observed in the presence of 0.1 mg/mL tRNA with a mean K_(D)of 42±12 nM. An example plot of ARC17480 binding to TFPI in both thepresence and absence of tRNA is shown in FIG. 12B.

Example 3

This example demonstrates that unlabeled ARC17480, ARC19498, ARC19499,ARC26835, ARC19500, ARC19501, ARC31301, ARC18546, ARC19881 and ARC19882compete with radiolabeled ARC17480 for binding to TFPI. This examplealso demonstrates that all of these aptamers have affinities for TFPIthat are similar to that observed with ARC17480.

Each aptamer was evaluated for binding to TFPI in a binding-competitionassay. For these experiments, human TFPI (American Diagnostica,Stamford, Conn., catalog #4500PC) was incubated with trace amounts ofradiolabeled ARC17480 and different concentrations of unlabeledcompetitor aptamer (5000 nM-0.25 nM for all aptamers except ARC19499;1000 nM-0.05 nM for ARC19499). For experiments with ARC17480 andARC19499 in FIG. 13A, 60 nM TFPI was used. For all other experiments(FIG. 13B-E), 10 nM TFPI was used. ARC17480 was included as a competitorin every experiment as a control. For each aptamer, the percentage ofradiolabeled ARC17480 bound at each competitor aptamer concentration wasused for analysis. The percentage of radiolabeled ARC17480 bound wasplotted as a function of aptamer concentration and fit to the equationy=(max/(1+x/IC₅₀))+int, where y=the percentage of radiolabeled ARC17480bound, x=the concentration of aptamer, max=the maximum radiolabeledARC17480 bound, and int=the y-intercept, to generate an IC₅₀ value forbinding-competition. FIG. 13A-E shows graphs of competition experimentswith ARC17480, ARC19498, ARC19499, ARC26835, ARC19500, ARC19501,ARC31301, ARC18546, ARC19881 and ARC19882. These molecules all competedsimilarly with radiolabeled ARC17480 for binding to TFPI. Theseexperiments demonstrate that ARC17480, ARC19498, ARC19499, ARC26835,ARC19500, ARC19501, ARC31301, ARC18546, ARC19881 and ARC19882 all bindsimilarly to full-length TFPI.

Example 4

This example demonstrates that the TFPI aptamers bind specifically toTFPI.

In this experiment, ARC17480 was tested for binding to a variety ofproteins that are key molecules in the coagulation cascade, moleculeswhose inhibition would show a similar profile to TFPI inhibition, ormolecules that are similar in structure or function to TFPI. Proteinsinvestigated were TFPI, Factor Va (FVa), Factor XII (FXII), antithrombin(ATIII), heparin cofactor II (HCII), alpha-thrombin, prothrombin, FactorVIIa (FVIIa), Factor IXa (FIXa), Factor Xa (FXa), Factor XIa (FXIa),kallikrein, plasmin, alpha-1 antitrypsin (serpin-A1), TFPI-2, andGST-TFPI-2. In the presence of up to 0.5-1 μM of each protein, ARC17480did not have any significant affinity for the sequence- andmechanistically-related proteins tested (FIG. 14A-D). ARC17480 showedsome binding to FXII at higher concentrations of protein. This bindingwas eliminated in the presence of 0.1 mg/mL tRNA (FIG. 14D), indicatingthat the binding was likely non-specific.

These experiments indicate that any effects on coagulation that aremediated by the TFPI aptamers are likely due to direct binding andinhibition of TFPI.

Example 5

This example demonstrates that ARC19499 binds tightly to TFPI in aplate-based binding assay. This example also demonstrates that ARC19498binds tightly to TFPI due to its competition with ARC19499 for bindingto TFPI in a plate-based binding assay.

In order to assess the binding affinity of ARC19499 to TFPI, recombinanthuman TFPI protein (0.5 mg/mL) was diluted in Dulbecco'sPhosphate-buffered Saline (DPBS) to a final concentration of 15 μg/mL,and 100 μL was added to a 96-well Maxisorb plate and incubated overnightat 4° C. The TFPI solution was then removed and the plate wassubsequently washed 3 times with 200 μL wash buffer (DPBS+0.05% Tween20) at room temperature. The plate was then blocked with 200 μL of 10mg/mL bovine serum albumin (BSA) in DPBS for 30 minutes at roomtemperature. The BSA blocking solution was then removed and the platewas washed again 3 times with 200 μL wash buffer. Serially dilutedARC19499 in DPBS with 0.1% BSA was then added to the plate and incubatedfor 3 hours at room temperature. After washing 3 times with 200 μL washbuffer, 100 μL of 0.5 μg/mL rabbit monoclonal anti-PEG antibody(Epitomics) was added to the plate and incubated for 60 minutes at roomtemperature. The anti-PEG antibody solution was then removed and theplate was washed as described above. Then, 100 μL anti-rabbit IgG-HRPsecondary antibody (Cell Signaling Technology), diluted 1000-fold inassay buffer, was added to each well and incubated for 30 minutes. Afterwashing 3 times with 200 μL wash buffer, 100 μL of TMB solution (Pierce)was added to each well and incubated for 2 minutes before adding 100 μLstop solution (2N H₂SO₄) to each well to stop the reaction. The assayplate was then read at 450 nm using a Victor³V 1420 multilabel counter(Perkin Elmer). Five different binding experiments suggested that thebinding affinity between ARC19499 and recombinant TFPI is 30 nM in theplate-based binding assay. The data from one of these experiments isshown in FIG. 15.

In order to assess the affinity of ARC19498 towards TFPI protein, anARC19499:TFPI binding competition assay was set up. Recombinant humanTFPI protein (0.5 mg/mL) was diluted in DPBS to a final concentration of15 μg/mL, and 100 μL was added to a 96-well Maxisorb plate and incubatedovernight at 4° C. The TFPI solution was then removed and the plate wassubsequently washed 3 times with 200 μL wash buffer (DPBS+0.05% Tween20) at room temperature. The plate was then blocked with 200 μL of 10mg/mL BSA in DPBS for 30 minutes at room temperature. The BSA blockingsolution was then removed and the plate was washed again 3 times with200 μL wash buffer. ARC19498 was serially diluted and mixed at differentconcentrations with 20 nM ARC19499 in DPBS in 0.1% BSA. TheARC19498:ARC19499 mixtures were added to the plate and incubated for 3hours at room temperature. After washing 3 times with 200 μL washbuffer, 100 μL of 0.5 μg/mL rabbit monoclonal anti-PEG antibody(Epitomics) was added to the plate and incubated for 60 minutes at roomtemperature. The anti-PEG antibody solution was then removed and theplate was washed as described above. Then, 100 μL anti-rabbit IgG-HRPsecondary antibody (Cell Signaling Technology), diluted 1000-fold inassay buffer, was added to each well and incubated for 30 minutes. Afterwashing 3 times with 200 μL wash buffer, 100 μL of TMB solution (Pierce)was added to each well and incubated for 2 minutes before adding 100 μLstop solution (2N H₂SO₄) to each well to stop the reaction. The assayplate was then read at 450 nm using a Victor³V 1420 multilabel counter(Perkin Elmer). The percent inhibition of ARC19499 binding wascalculated using 0 nM ARC19498 in 20 nM ARC19499 as 0% inhibition, and 0nM ARC19498 and 0 nM ARC19499 as 100% inhibition. The IC₅₀ wascalculated based on 4-parameter logistics using Graphpad Prism 4software. FIG. 16 shows two replicates of this experiment, both of whichgave an IC₅₀ of 20 nM for ARC19498 competition with ARC19499 in thisassay. These results suggest that ARC19498 has a binding affinity forTFPI in the plate-based binding assay that is similar to that observedfor ARC19499.

Example 6

This example examines the regions on TFPI where ARC17480 binds. Dot blotbinding experiments were carried out with radiolabeled ARC17480 andvarious truncated TFPI proteins, and binding-competition experimentswere carried out with radiolabeled ARC17480, TFPI, and heparin or lowmolecular weight heparin (LMWH). The proteins used for bindingexperiments are described in Table 1 below.

Trace amounts of radiolabeled ARC17480 were incubated with differentconcentrations (500 nM-0.7 nM) of full-length TFPI and TFPI-His (Table1). FIG. 17A shows that ARC17480 had reduced binding to TFPI-His whencompared to its binding to full-length TFPI. This experiment suggestedthat the C-terminal 20 amino acids of TFPI, which are missing inTFPI-His but present in full-length TFPI, contribute to binding ofARC17480 to TFPI.

Trace amounts of radiolabeled ARC17480 were incubated with differentconcentrations of truncated TFPI-K1K2 (500 nM-0.008 nM) and theK3-C-terminal domain protein (500 nM-0.7 nM) (Table 1). FIG. 17B showsthat ARC17480 had no detectable binding to truncated TFPI-K1K2 and veryweak binding to the K3-C-terminal domain protein that was onlydetectable at higher concentrations of the protein. Trace amounts ofradiolabeled ARC17480 were incubated with different concentrations ofthe C-terminal peptide (10 μM-0.17 nM) (Table 1). Neutravidin (˜100 nMmonomer) was then added to the binding solution to assist in the captureof aptamer:peptide complexes on a nitrocellulose filter. The amount ofradiolabeled aptamer captured on a nitrocellulose filter was quantitatedand compared to the total amount of radiolabeled aptamer to generate abinding curve, which is shown in FIG. 17B. ARC17480 showed weak bindingto the C-terminal peptide at high concentrations of peptide.

Trace amounts of radiolabeled ARC17480 were incubated with differentconcentrations of full-length TFPI (500 nM-0.008 nM) in the absence orpresence of 0.1 mg/mL unfractionated heparin (FIG. 18A). The inclusionof heparin in the binding experiment completely abolished ARC17480binding to TFPI. In a separate experiment, trace amounts of radiolabeledARC17480 were incubated with 12.5 nM full-length TFPI and differentconcentrations (5 μM-0.25 nM) of unfractionated heparin or low molecularweight heparin (LMWH). FIG. 18B shows that both unfractionated heparinand LMWH competed with ARC17480 for binding to TFPI in aconcentration-dependent manner. Heparin was a more effective competitorthan LMWH. The K3-C-terminal regions of TFPI have been implicated inglycocalyx binding, and this is the region of the protein where heparinand LMWH should bind. These experiments suggest that the K3-C-terminalregion of TFPI is important for ARC17480 binding to TFPI.

Taken together, these experiments demonstrate that the C-terminal domainlikely participates in ARC17480 binding to TFPI. These experiments alsodemonstrate that the ARC17480 binding region on TFPI is not entirelycontained within the K1-K2 region of the protein, or within theK3-C-terminal region of the protein. The regions required for ARC17480binding likely spans more than one domain of the protein.

TABLE 1 Proteins used for binding experiments Amino acids of ProteinDescription mature TFPI Full-length American Diagnostica, 1-276 TFPI E.coli expressed TFPI-His R&D systems, murine myeloma 1-256 + C-terminalcell line expressed 10-His tag Truncated American Diagnostica, 1-161TFPI-K1K2 E. coli expressed K3-C-terminal E. coli expressed N-terminal6-His domain tag + 182-276 C-terminal synthetic N-terminal biotin +peptide 242-276

Example 7

This example examines the regions on TFPI where ARC17480 and ARC19499bind. For these experiments, antibodies that bind to different regionson TFPI were used to compete for binding to TFPI with ARC19499 in aplate-based binding assay, or to compete for binding to TFPI withARC17480 in a dot-blot binding assay. The antibodies used forcompetition are shown in Table 2 below.

This example demonstrates that antibody AD4903 (American Diagnostica,catalog #4903) competed for binding of ARC19499 to TFPI in a plate-basedbinding assay and competed for binding of ARC17480 to TFPI in a dotblot-binding assay (FIG. 19A and FIG. 20C). Antibody AD4903 was raisedagainst a fragment of TFPI containing K1 domain amino acid residues22-87, and binds to TFPI somewhere in this region (Table 2). Thisexample also demonstrates that antibody ACJK-4, which was raised againsta peptide that contained amino acid residues 148-162 that are part ofthe intervening region between the K2 and K3 domains of TFPI, competedweakly with ARC17480 for binding to TFPI in a dot-blot binding assay(FIG. 20B). This example also demonstrates that antibodies ACJK-1 andACJK-2, which were raised against peptides that contained amino acidresidues 261-276 and 245-262, respectively, that are part of theC-terminal domain of TFPI, partially competed for ARC19499 binding toTFPI in a plate-based binding assay (FIG. 19B). This example furtherdemonstrates that several other antibodies that bind to differentregions of TFPI did not compete with ARC19499 binding to TFPI in aplate-based binding assay, and did not compete with ARC17480 for bindingto TFPI in a dot blot-based binding assay. The antibodies used for thecompetition experiments are shown in Table 2 below.

For plate-based binding experiments, 400 ng/well of TFPI (AmericanDiagnostics, cat# 4900PC) in 100 μL Dulbecco's Phosphate-buffered Saline(DPBS) was used to coat a 96-well Maxisorb plate at 4° C. The TFPIsolution was then removed and the plate was subsequently washed 3 timeswith 200 μL wash buffer (DPBS+0.05% Tween 20) at room temperature. Theplate was then blocked with 200 μL of 10 mg/mL bovine serum albumin(BSA) in DPBS for 30 minutes at room temperature. The BSA blockingsolution was then removed and the plate was washed 3 times with 200 μLwash buffer. The competing antibodies were serially diluted and mixedwith ARC19499 at the final concentration of 25 nM ARC19499 and 0.1% BSAin DPBS, and the mixture was then added to the assay plate and incubatedfor 3 hours at room temperature. ARC19498 was similarly mixed withARC19499 and used as a positive control in the antibody competitionassay. Wells were then washed, as described above. For experiments usingantibodies AD4903, AD4904 and 7035-A01 for competition, 100 μL of 0.5μg/mL rabbit monoclonal anti-PEG antibody (Epitomics, cat #2061-1) inassay buffer was added to the plate and incubated for 3 hours at roomtemperature. Anti-PEG antibody solution was then removed and the platewas washed as described above, followed by addition of 100 μL of1:1000-diluted anti-rabbit IgG-HRP secondary antibody in assay buffer toeach well (Cell Signaling Technology, cat #7074) and incubated for 30minutes. The secondary antibody solution was removed and the plate waswashed, as described above. For antibodies ACJK1-ACJK5, 0.5 μg/mL of 100μL biotinylated rabbit monoclonal anti-PEG antibody (Epitomics, cat#2173) in assay buffer was added to the assay plate and incubated for 3hours at room temperature, followed by washing, as described above. Theantibody was then removed and the plate was washed as described above,followed by addition of 100 μL of streptavidin-HRP (4800-30-06) from R&DSystems (Minneapolis, Minn.) diluted 200-fold in DPBS and incubated foran additional 1 hour at room temperature. The streptavidin-HRP was thenremoved and the plate was washed as described above. Then 100 μL of TMBsolution (Pierce, #34028) solution was added to each well and incubatedfor 2 minutes, followed by addition of 100 μL stop solution (2N H₂SO₄)to each well to stop the reaction. The assay plate was then read at 450nm using a Victor³V 1420 multilabel counter (Perkin Elmer). Percentinhibition of binding was calculated using 0 nM antibody in 25 nMARC19499 as 0% inhibition, and 0 nM antibody and 0 nM ARC19499 as 100%inhibition. The IC_(so) was calculated based on 4-parameter logisticsusing Prism 4 Graphpad software.

As shown in FIG. 19A, antibody AD4903, which binds within the K1 domainof TFPI, competed with ARC19499 for binding to recombinant TFPI in theplate-based binding assay. Antibodies ACJK-1 and ACJK-2, which bind toregions within the C-terminal region of TFPI, partially competed forARC19499 binding to TFPI in the plate-based binding assay (FIG. 19B).Antibodies AD4904, ACJK-3, ACJK-4, ACJK-5 and 7035-A01 showed nocompetition for ARC19499 binding to TFPI in the plate-based bindingassay (FIGS. 19A and B). These experiments suggest that the K1 regionand the C-terminal region of TFPI are involved in ARC19499 binding toTFPI.

The antibodies in Table 2 were also tested in a dot blot-basedcompetition binding assay. In these experiments, trace amounts ofradiolabeled ARC17480 were incubated with 10 nM recombinant TFPI, withor without the addition of antibody. Antibodies were tested at 1000 nM,333 nM, 111 nM, 37.0 nM, 12.4 nM, 4.12 nM, 1.37 nM, 0.46 nM, 0.15 nM and0.051 nM. ARC17480 was included as a competitor in every experiment as acontrol. For each molecule, the percentage of radiolabeled ARC17480bound at each competitor aptamer concentration was used for analysis.The percentage of radiolabeled ARC17480 bound was plotted as a functionof aptamer concentration and fit to the equation y=(max/(1+x/IC₅₀))+int,where y=the percentage of radiolabeled ARC17480 bound, x=theconcentration of aptamer, max=the maximum radiolabeled ARC17480 bound,and int=the y-intercept, to generate an IC₅₀ value forbinding-competition. FIG. 20 shows the binding-competition experimentscarried out with ACJK-1, ACJK-2, ACJK-3, ACJK-4, ACJK-5, AD4903 andAD4904. These experiments demonstrate that antibody AD4903 competed forARC17480 binding to TFPI in the dot blot competition assay (FIG. 20C).ACJK-4 partially competed for binding in this assay (FIG. 20B), whileACJK-1, ACJK-2, ACJK-3, ACJK-5 and AD4904 showed no significantcompetition for binding with ARC17480 (FIG. 20A-C). These experimentssuggest that the K1 region and the K2-K3 intervening regions of TFPI areinvolved in ARC17480 binding to TFPI.

Taken together, these experiments demonstrate that the K1 region of TFPIis likely involved in ARC17480/ARC19499 binding to TFPI. Theseexperiments also suggest that the C-terminal and the K2-K3 interveningregions of TFPI may be involved in aptamer binding. Lack of bindingcompetition of antibodies to other regions of TFPI does not precludetheir involvement in aptamer binding.

TABLE 2 Antibodies used in ARC19499 competition assays Region of matureTFPI used as an antigen for Antibody target Antibody Type antibodygeneration region in TFPI AD4903 mouse 22-87 K1 region monoclonal AD4904mouse  88-160 K2 region monoclonal ACJK-1 rabbit 261-276 C-terminalpeptide polyclonal ACJK-2 rabbit 245-262 C-terminal peptide polyclonalACJK-3 rabbit 192-204 K3 peptide polyclonal ACJK-4 rabbit 148-162 K2-K3intervening polyclonal peptide ACJK-5 rabbit 54-69 K1 peptide polyclonal7035-A01 mouse 152-252 K3-C-terminal polyclonal region

Example 8

This example demonstrates that ARC19499 has in vitro activity inhibitingTFPI in the extrinsic tenase (Xase) inhibition assay.

In this assay, tissue factor (TF) was mixed with Factor VIIa (FVIIa) andphospholipid vesicles. Factor X (FX) was added and aliquots were removedand quenched at various time points. At this point, a chromogenicsubstrate for Factor Xa (FXa) was added and absorbance at 405 nm wasmeasured over a course of an hour in order to determine rates of FXageneration. When 1 nM TFPI was included, the rate of FXa generation wassignificantly decreased. This was seen in FIG. 21A when comparing thefilled diamonds (no TFPI) with the empty circles (1 nM TFPI). Whenincreasing concentrations of ARC19499 were also included along with the1 nM TFPI, there was a dose-dependent improvement on the rate of FXageneration. 1000 nM ARC19499 (empty diamonds) resulted in rates of FXageneration that were close to the rate of FXa generation in the absenceof TFPI (filled diamonds) (FIG. 21A). These rates were normalized bydividing the rate at one specific time point (in this case, at 4minutes) by the rate achieved with no TFPI at that same time point (FIG.21B). In this manner, TFPI reduced the rate of FXa generation at 4minutes by nearly 70%. Increasing concentrations of ARC19499 improvedthis rate, reaching levels close to that of no TFPI with 10-1000 nMaptamer (FIG. 21B).

This experiment indicates that ARC19499 inhibits TFPI in an in vitroextrinsic tenase inhibition assay.

Example 9

This example demonstrates that ARC26835, ARC17480, ARC19498, ARC19499,ARC19500, ARC19501, ARC31301, ARC18546, ARC19881 and ARC19882 haveTFPI-inhibitory activity in the Factor Xa (FXa) activity assay.

Each aptamer was evaluated for inhibition of TFPI in a Factor Xa (FXa)activity assay. The ability of FXa to cleave a chromogenic substrate wasmeasured in the presence and absence of TFPI, with or without theaddition of aptamer. For these experiments, 2 nM human FXa was incubatedwith 8 nM human TFPI. Then, 500 μM chromogenic substrate and aptamerswere added, and FXa cleavage of the substrate was measured by absorbanceat 405 nm (A₄₀₅) as a function of time. Aptamers were tested at 500 nM,125 nM, 31.25 nM, 7.81 nM, 1.95 nM and 0.49 nM concentrations. ARC17480was included as a control in each experiment. For each aptamerconcentration, the A₄₀₅ was plotted as a function of time and the linearregion of each curve was fit to the equation y=mx+b, where y=A₄₀₅, x=theaptamer concentration, m=the rate of substrate cleavage, and b=they-intercept, to generate a rate of FXa substrate cleavage. The rate ofFXa substrate cleavage in the presence of TFPI and the absence ofaptamer was subtracted from the corresponding value in the presence ofboth TFPI and aptamer for each aptamer at each concentration. Then, theadjusted rates were plotted as a function of aptamer concentration andfit to the equation y=(V_(max)/(1+IC₅₀/x)), where y=the rate ofsubstrate cleavage, x=concentration of aptamer, and V_(max) the maximumrate of substrate cleavage, to generate an IC₅₀ and maximum (V_(max))value. FIG. 22A-C show graphs of FXa activity assays with ARC26835,ARC17480, ARC19498, ARC19499, ARC19500, ARC19501, ARC31301, ARC18546,ARC19881 and ARC19882. These aptamers all inhibited TFPI in theseassays, as evidenced by an increase in FXa activity as a function ofaptamer concentration. These aptamers all had similar activity in theFXa assay.

Example 10

This example demonstrates that ARC19499 protects Factor Xa (FXa) frominhibition by TFPI in a chromogenic assay with purified components.

FXa (1 nM), TFPI (2.5 nM), ARC19499 (0-500 nM) and Spectrozyme Xa(American Diagnostica) chromogenic substrate (200 μM) were incubated inHEPES-buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4) containing 2 mMCaCl₂ and 0.1% PEG-6,000 (HBSP2 buffer) at 37° C. until equilibrium wasachieved (5 minutes). The rate of Spectrozyme FXa hydrolysis wasdetermined using a ThermoMax instrument (Molecular Devices) and plottedas the % FXa activity compared to no TFPI (100%). Increasingconcentrations of ARC19499 caused an increase in FXa activity (FIG. 23),demonstrating that ARC19499 protected FXa from the inhibition by TFPI.Based on this data, the apparent dissociation constant (K_(D)) ofARC19499 for TFPI was 1.8 nM. ARC19499 is specific for TFPI and did notinhibit FXa activity in the absence of TFPI (data not shown).

Example 11

This example demonstrates that ARC19499 protects the extrinsic FXasecomplex, which is composed of tissue factor, Factor VIIa (FVIIa) andFactor Xa (FXa), from inhibition by TFPI in a chromogenic activity assaywith purified components.

Relipidated tissue factor (TF; 20 pM), FVIIa (1 nM), PCPS vesicles (75%phosphatidyl choline/25% phosphatidyl serine; 20 μM) and ARC19499(0-1000 nM) were incubated in HBSP2 buffer at 37° C. for 10 minutes,followed by the simultaneous addition of FX (1 μM) and TFPI (2.5 nM).Aliquots were removed every 30 seconds for 5 minutes and quenched intoHBS buffer containing 20 mM EDTA and 0.1% PEG. Spectrozyme FXa substrate(200 μM) was added, the rate of substrate hydrolysis was measured, andthe concentration of active FXa was estimated from a calibration curve.Increasing concentrations of ARC19499 caused an increase in FXa activity(FIG. 24) up to 70% of the rate measured in the absence of TFPI,demonstrating that ARC19499 substantially protected the extrinsic FXasecomplex from the inhibition by TFPI.

Example 12

This example demonstrates that ARC19499 protects the tissue factor:FVIIacomplex from TFPI inhibition in a fluorogenic assay of tissuefactor:FVIIa activity carried out with purified components.

Tissue factor (TF; 1 nM), FVIIa (2 nM) and ARC19499 (0-7.5 nM) wereincubated in HBSP2 at 37° C. for 10 minutes, followed by thesimultaneous addition of a fluorogenic substrate SN-17c (50 μM) and TFPI(8 nM). The rate of substrate hydrolysis was measured in a fluorescenceplate reader (BioTek). TFPI inhibited approximately 50% of TF:FVIIaactivity under these conditions (FIG. 25). The addition of astoichiometric concentration of ARC19499 (8 nM) completely restored fullTF:FVIIa activity compared to a no TFPI control (FIG. 25), demonstratingthat ARC19499 efficiently protected the TF:FVIIa complex from TFPIinhibition. A titration of increasing ARC19499 concentrations in thepresence of 8 nM TFPI increased the activity of the TF:FVIIa complex inan ARC19499 concentration-dependent manner, reaching the mid-point ofactivity at ˜1 nM ARC19499. Data analysis indicated that the apparentK_(D) of ARC19499 for TFPI in this assay was 1.2 nM. ARC19499 isspecific for TFPI and did not inhibit TF:FVIIa activity in the absenceof TFPI (data not shown).

Example 13

This example demonstrates that ARC19499 inhibits TFPI in a syntheticcoagulation proteome that models hemophilia A and hemophilia B. Thesedata show that ARC19499 restored normal thrombin generation in thepresence of complete (0%) Factor VIII (FVIII) or Factor IX (FIX)deficiency. ARC19499 also restored normal thrombin generation in thepresence of incomplete (2%, 5% or 40%) FVIII deficiency.

Thrombin generation was initiated with 5 μM relipidated tissue factor(TF) added to a mixture of procoagulants and coagulation inhibitors(Factors V, VII, VIIa, VIII, IX, X, XI, prothrombin, antithrombin andTFPI; all at mean physiologic concentrations) and 50 μM PCPS (75%phosphatidyl choline/25% phosphatidyl serine). Thrombin generation overtime was measured in a chromogenic assay using the Spectrozyme THsubstrate (American Diagnostica). ARC19499 was tested at increasingconcentrations of 1 nM, 2.5 nM, 5 nM and 10 nM in a fully reconstitutedsystem (healthy control) or in reconstituted systems in which eitherFVIII (severe hemophilia A) or FIX (severe hemophilia B) was omitted.

In the presence of all proteins at their mean physiologic concentrations(“healthy control”; FIG. 26), the initiation (lag) phase of thrombingeneration initiated with 5 pM relipidated TF was approximately 6minutes, and the maximum concentration of active thrombin observed was270 nM (filled diamonds). The omission of TFPI significantly shortenedthe initiation phase (to 2 minutes) and increased maximum thrombinconcentration to 374 nM (filled circles). Additions of increasingARC19499 concentrations in the presence of 2.5 nM TFPI decreased theduration of the initiation phase and increased maximum thrombinconcentration in an ARC19499-dependent manner, and at 10 nM (emptysquares) the ARC19499 thrombin generation profile was almost identicalto that observed in the absence of both TFPI and ARC19499 (FIG. 26).

In the absence of FVIII, TF initiated thrombin generation wassignificantly suppressed (FIG. 27). The initiation phase was extendedfrom 6 minutes in the “healthy control” (filled diamonds) to 10 minutesin hemophilia A (empty diamonds), and maximum thrombin activitydecreased from 270 nM to 34 nM. The omission of TFPI in the absence ofFVIII restored normal thrombin generation (maximum concentration ofactive thrombin increases to 264 nM) and the duration of the initiationphase decreased to 2 minutes (filled circles). In the absence of FVIIIand in the presence of 2.5 nM TFPI, the addition of 5 nM ARC19499(asterisks) restored thrombin generation to the level observed in the“healthy control” with the initiation phase of 3 minutes. In thepresence of 10 nM ARC19499 (empty squares) and 2.5 nM TFPI and in theabsence of FVIII, the thrombin generation profile became similar to thatobserved in the absence of TFPI, FVIII and ARC19499.

The effect of TFPI inhibition by ARC19499 in a hemophilia B (no FIX)synthetic coagulation proteome was similar to that observed for thehemophilia A model, i.e., ARC19499 at 5 nM concentration and in theabsence of FIX restored thrombin generation to the level similar to thatobserved in “healthy control” (FIG. 28).

The effect of ARC19499 on TF-initiated thrombin generation was evaluatedin the synthetic coagulation proteome model at 0%, 2% (0.014 nM), 5%(0.035 nM), 40% (0.28 nM) and 100% (0.7 nM) FVIII. The selected FVIIIconcentrations covered the range observed in severe (<1%), moderate(1-5%) and mild (5-40%) hemophilia A patients. In the absence ofARC19499 (FIG. 29), thrombin generation was suppressed at all FVIIIconcentrations tested, up to and including 40%. The peak thrombin levelobserved in the 40% FVIII proteome (asterisks) was approximately 50% ofthe “healthy control” (filled diamonds). The addition of ARC19499 at aconcentration of 1 nM was sufficient to significantly boost thrombingeneration by shortening the initiation phase and increasing peakthrombin levels (FIG. 30). The addition of 2.5 nM ARC19499 essentiallynormalized thrombin generation in the presence of 0-5% FVIII (FIG. 31),while at 40% and 100% FVIII, 2.5 nM ARC19499 induced further shorteningof the initiation phase and increased peak thrombin, nearly to theextent of the “No TFPI” control.

FIG. 32 shows additional synthetic coagulation proteome data for 0%FVIII in the presence of a series of ARC19499 concentrations (0, 1, 2.5,5 and 10 nM) compared to a “healthy control” and a “No TFPI” control.FIG. 33 shows the data for 100% FVIII for the same range of ARC19499concentrations. FIGS. 34, 35 and 36 show the data for 2%, 5% and 40%FVIII in the presence of 0, 1 and 2.5 nM ARC19499, respectively. Underall conditions, ARC19499 showed a significant procoagulant response,causing the initiation phase (lag time) to decrease and the peakthrombin to increase. In all cases of FVIII deficiency (0-40% FVIII),ARC19499 was able to restore a normal thrombin generation profile.

Example 14

This example demonstrates that in vitro activity of ARC19499 is specificfor the presence of TFPI.

In this experiment, the ability of ARC19499 to affect thrombingeneration in the calibrated automated thrombogram (CAT) assay, whichmeasures the generation of thrombin over time following initiation ofthe tissue factor coagulation pathway, was tested in three differentplasma conditions. In the first condition, increasing concentrations ofARC19499 were added to pooled normal plasma (PNP) and mixed with asolution containing tissue factor (TF) and phospholipids so that the TFconcentration was either 0.1 or 1.0 μM in the final reaction volume(FIG. 37). Thrombin generation was initiated by the addition of amixture containing calcium chloride and a fluorogenic substrate forthrombin. The reaction took place at 37° C., and fluorescence intensitywas measured periodically over 1 hour. ARC19499 was tested at thefollowing concentrations in the plasma: 0.1, 1, 10, 100 and 1000 nM.

With either TF concentration, increasing ARC19499 increased thegeneration of thrombin in PNP plasma (FIG. 37A-B). Both the endogenousthrombin potential (ETP—area under the curve) and peak thrombin (highestlevel of thrombin produced at any one point in the assay) valuesincreased in a dose-dependent manner with ARC19499 (FIG. 37C-D). The lagtime (time it takes for thrombin generation to begin) decreased in adose-dependent manner with ARC19499 (FIG. 37E). These results wereobserved at both concentrations of TF.

The CAT assay measuring ARC19499 activity was repeated in TFPI-depletedplasma. Plasma that was immunodepleted for TFPI and lyophilized wasobtained from American Diagnostica (Stamford, Conn.) and resuspendedprior to use. Thrombin generation was measured as described above with0.01, 0.1 or 1.0 pM TF. The results in FIG. 38A show that the thrombingeneration curves measured for each TF concentration were distinct fromeach other, but within a specific TF concentration there was essentiallyno difference in thrombin generation as the ARC19499 concentration wasincreased. This was also seen in the parameters measured in the CATassay. There was little or no change in ETP, peak thrombin or lag timeas the ARC19499 concentration increased (FIG. 38B-D), independent of TFconcentration. ARC19499 activity was tested in a third set of plasmaconditions. In this case, PNP was incubated with a polyclonal antibodyagainst TFPI, in order to neutralize all TFPI activity. ARC19499 wasthen added to this antibody-treated plasma (FIG. 39). Again, thrombingeneration was initiated with either 0.01, 0.1 or 1.0 pM TF. Addition ofthe polyclonal antibody enhanced thrombin generation at all three TFconcentrations because the TFPI was neutralized; however, increasingconcentrations of ARC19499 appeared to cause no further increases inthrombin generation (FIG. 39A-C). There was little to no effect on theETP, peak thrombin or lag time when ARC19499 was added (FIG. 39D-F).

These experiments indicate that ARC19499 only has procoagulant activitywhen functioning TFPI is present in the plasma, and therefore, ARC19499is specific for TFPI.

Example 15

This example demonstrates that ARC17480, ARC19498 and ARC19499 inhibitTFPI activity in vitro.

In this experiment, the inhibitory activity of TFPI aptamers (ARC17480,ARC19498 and ARC19499) were measured in vitro in pooled hemophilia A(Factor VIII-deficient) plasma in a calibrated automated thrombogram(CAT) assay. Aptamers were titrated at different concentrations inpooled hemophilia A plasma, and the amount of thrombin generated wascompared to a pooled normal plasma control (FIG. 40A-C), using 1.0 pM TFin the final reaction. Both the endogenous thrombin potential (ETP),which is the area under the thrombin generation versus time curve, andthe peak thrombin, which is the highest concentration of thrombingenerated over the course of the experiment, provided indirect measuresof aptamer inhibition of TFPI. All three aptamers had similar activityin this assay, with ARC19499 having slightly higher activity than theother two. ARC19499 corrected the ETP to near normal levels by 30 nM(FIG. 40D). Peak thrombin levels also increased with increasingconcentrations of aptamer (FIG. 40E).

These results show that ARC17480, ARC19498 and ARC19499 inhibit TFPIactivity in vitro.

Example 16

This example demonstrates that ARC19499 increases thrombin generation innormal human plasma treated with an anti-Factor VIII antibody togenerate a hemophilia A-like state.

Platelet-poor plasma from a normal, healthy volunteer was treated withan anti-FVIII antibody to generate a hemophilia A-like state. Thrombingeneration in this antibody-treated plasma was similar to that observedwith hemophilia A plasma (FIG. 41). Addition of ARC19499 to theantibody-treated plasma resulted in a dose-dependent increase inthrombin generation. These results demonstrate that ARC19499 can correctthrombin generation in plasma with low FVIII levels that results fromtreatment with an anti-FVIII antibody.

Example 17

This example demonstrates that ARC17480 and ARC19499 inhibit TFPIactivity in vitro and have biological activity.

The effects of the non-PEGylated core TFPI inhibitory aptamer ARC17480and the PEGylated aptamer ARC19499 were evaluated for in vitro thrombingeneration activity in hemophilia B (FIX-deficient) plasma using thecalibrated automated thrombogram (CAT) assay.

These studies were performed in plasma pooled from two hemophilia Bpatients with <1% Factor IX levels (commercially available from GeorgeKing Bio-Medical, Inc, Overland Park, Kans.). In this assay, plasma andaptamer were mixed together and added to a reagent containingphospholipids and tissue factor. Thrombin generation was initiated bythe addition of a mixture containing calcium chloride and a fluorogenicsubstrate for thrombin. The reaction took place at 37° C., andfluorescence intensity was measured periodically over 1 hour. The finalconcentrations of tissue factor and phospholipids were 1 pM and 4 μM,respectively. Aptamers were tested at the following concentrations inthe plasma: 0.3, 1, 3, 10, 30, 100, 300 and 1000 nM. Individual thrombingeneration curves are shown in FIG. 42, illustrating the effects ofincreasing concentrations of ARC19499 (FIG. 42A) or ARC17480 (FIG. 42B)on the extent of thrombin generation in hemophilia B plasma compared topooled normal plasma. The plot of ETP, peak thrombin and lag time areshown in FIG. 43. Results with ARC19499 are plotted on the left side,and results with ARC17480 are plotted on the right side.

The ETP and peak thrombin levels decreased ˜85% and ˜95%, respectively,in plasma from the hemophilia B pool compared to the pooled normalplasma, consistent with a deficiency in thrombin generation due to theloss of Factor IX. Both ARC17480 (triangles) and ARC19499 (diamonds)largely corrected the defect in thrombin generation, as measured by bothof these parameters (FIG. 43). By 100 nM, both aptamers demonstrated anETP nearly equivalent to that achieved with pooled normal plasma, and anearly equivalent peak thrombin by 300 nM ARC17480 (FIG. 43). Peakthrombin plateaued by 300 nM ARC19499. ARC17480 and ARC19499 decreasedthe lag time in hemophilia B plasma, below what was achieved with poolednormal plasma and hemophilia B plasma without any drug (FIG. 43).

These results show that ARC17480 and ARC19499 inhibit TFPI with similarpotency in hemophilia B plasma in vitro.

Example 18

This example demonstrates that ARC19499 inhibits TFPI activity in vitroand has biological activity compared to a negative control aptamer.

The ability of ARC19499 to enhance thrombin generation was tested inthree platelet-poor hemophilia plasmas: plasma pooled from 7-8 patientswith severe hemophilia A (<1% FVIII levels; referred to as “hemophilia Aplasma”), plasma from three different hemophilia A patients with hightiters of anti-FVIII antibodies (≧160 Bethesda units (BU)/mL; referredto as “inhibitor plasma”), and plasma pooled from two patients withsevere hemophilia B (<1% FIX levels; referred to as “hemophilia Bplasma”). All plasmas were from George King Bio-Medical (Overland Park,Kans.). Thrombin generation was measured using the calibrated automatedthrombogram (CAT) assay. In this assay, plasma and aptamer were mixedtogether and added to a reagent containing phospholipids and tissuefactor. Thrombin generation was initiated by the addition of a mixturecontaining calcium chloride and a fluorogenic substrate for thrombin.The reaction took place at 37° C., and fluorescence intensity wasmeasured periodically over 1 hour. The final concentrations of tissuefactor and phospholipids were 1 pM and 4 μM, respectively. Thrombingeneration in the presence of ARC19499 (0.3, 1, 3, 10, 30, 100, 300 and1000 nM) was compared to negative control aptamer (0.1, 1, 10, 100 and1000 nM). Plots of ETP, peak thrombin and lag time (mean±s.e.m.) areshown in FIG. 44.

Hemophilia A plasma had a slightly shorter lag time and a markedlydecreased ETP and peak thrombin (˜50% and ˜75%, respectively) comparedto normal plasma. Increasing concentrations of ARC19499 largelycorrected the defect in thrombin generation. ETP was corrected tonear-normal levels with 3 nM ARC19499, and peak thrombin was correctedwith 100 nM aptamer (FIG. 44A) Inhibitor plasma also had decreased ETPand peak thrombin (˜50% and ˜70%, respectively) compared to normalplasma. As with the severe hemophilia A plasma, ARC19499 increasedthrombin generation in this plasma. With 30 nM ARC19499, both the ETPand peak thrombin were at normal levels (FIG. 44B). Hemophilia B had aneven greater defect in thrombin generation, with significantly decreasedETP and peak thrombin (˜70% and ˜90%, respectively) and an increased lagtime. As with the hemophilia A plasmas, increasing concentrations ofARC19499 improved thrombin generation in this plasma, achieving normalETP levels with 30 nM ARC19499, and normal peak thrombin levels with100-300 nM aptamer (FIG. 44C). Taken together, these results demonstratethat 30-100 nM ARC19499 is effective in restoring coagulation in threedifferent types of hemophilia plasma. A negative control aptamer wasalso tested in the three different plasmas and demonstrated nocorrection of thrombin generation (FIG. 44).

The sequence of the negative control aptamer, ARC32603, used in thisexample was:mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-mG-dC-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 152).

Example 19

This example demonstrates that ARC17480, ARC26835, ARC19500, ARC19501,ARC31301, ARC18546, ARC19881 and ARC19882 have biological activity inthe calibrated automated thrombogram (CAT) assay.

The TFPI-inhibitory activity of each aptamer was evaluated in the CATassay in pooled hemophilia A plasma at 500 nM, 167 nM, 55.6 nM, 18.5 nM,6.17 nM and 2.08 nM aptamer concentration. ARC17480 was included inevery experiment as a control. For each aptamer, the endogenous thrombinpotential (ETP) and peak thrombin values at each aptamer concentrationwere used for analysis. The ETP or peak thrombin value for hemophilia Aplasma alone was subtracted from the corresponding value in the presenceof aptamer for each molecule at each concentration. Then, the adjustedETP and peak values were plotted as a function of aptamer concentrationand fit to the equation y=(max/(1+IC₅₀/x))+int, where y=ETP or peakthrombin, x=concentration of aptamer, max=the maximum ETP or peakthrombin, and int=the y-intercept, to generate an IC₅₀ value for boththe ETP and the peak thrombin. FIG. 45A-D and FIG. 46A-B show graphs ofCAT experiments with ARC17480, ARC26835, ARC19500, ARC19501, ARC31301,ARC18546, ARC19881 and ARC19882. Both the adjusted endogenous thrombinpotential (ETP) and peak thrombin are shown. These experimentsdemonstrate that ARC17480, ARC26835, ARC19500, ARC19501, ARC31301,ARC18546, ARC19881 and ARC19882 all functionally inhibited TFPI in theCAT assay, as evidenced by a concentration-dependent increase in bothETP and peak thrombin in hemophilia A plasma. These molecules all havesimilar activity in the CAT assay.

Example 20

This example demonstrates that the TFPI aptamers have biologicalactivity.

In this experiment, the ability of ARC19499 to affect thrombingeneration compared to that of NovoSeven® was tested using thecalibrated automated thrombogram (CAT) assay. The CAT assay generates anumber of parameters to compare thrombin generation. The lag time is ameasure of the length of time that it takes for thrombin generation tobegin. Peak thrombin is a measure of the highest amount of thrombin tobe generated at any one point. The endogenous thrombin potential (ETP)is the area under the thrombin generation curve.

These studies were performed in the presence of three different plasmas:platelet-poor plasma from healthy volunteers, a pool of plasma fromhemophilia A patients with <1% Factor VIII levels (commerciallyavailable from George King Bio-Medical, Inc, Overland Park, Kans.), andplasma from hemophilia A patients with a high titer of inhibitorantibody to Factor VIII (commercially available from George KingBio-Medical, Inc, Overland Park, Kans.). In the CAT assay, plasma anddrug (either ARC19499 or NovoSeven®) were mixed together and added to areagent containing phospholipids and tissue factor. Thrombin generationwas initiated by the addition of a mixture containing calcium chlorideand a fluorogenic substrate for thrombin. The reaction took place at 37°C., and fluorescence intensity was measured periodically over 1 hour.The final concentrations of tissue factor and phospholipids were 1 pMand 4 μM, respectively. The drugs were tested at the followingconcentrations in the plasma: 0.3, 1, 3, 10, 30, 100 and 300 nM.

In the plasma from healthy volunteers, there was no change in the ETPover the range of concentrations tested with both ARC19499 andNovoSeven® (FIG. 47A). The peak thrombin levels increased slightly atthe higher doses, with ARC19499 and NovoSeven® behaving in a nearlyidentical manner (FIG. 47B). ARC19499 had no effect on the lag time ofthrombin generation, while NovoSeven® demonstrated a dose-dependentdecrease in lag time, reaching a minimum lag time by 30 nM (FIG. 47C).

The ETP and peak thrombin levels decreased ˜40% and ˜75%, respectively,in plasma from the hemophilia A pool, consistent with a deficiency inthrombin generation due to the loss of Factor VIII. ARC19499 andNovoSeven® largely corrected the defect in thrombin generation, asmeasured by both of these parameters. These agents demonstrated a nearlyequivalent effect on ETP, reaching a maximum ETP by 30 nM (FIG. 48A).NovoSeven® had a slightly higher effect on peak thrombin reaching amaximal level by 30 nM. ARC19499 reached the same level of peak thrombinby 300 nM (FIG. 48B). As seen in the plasma from healthy volunteers,ARC19499 had no effect on the lag time, while NovoSeven® showed adose-dependent decrease in lag time, reaching a maximum effect by 30 nM(FIG. 48C).

Similar results were seen in the plasma from patients with a high titerantibody. Both drugs increased ETP and peak thrombin in the same manner(FIGS. 49A-B). Again, ARC19499 had no effect on the lag time, whileNovoSeven® showed a dose-dependent decrease of lag time (FIG. 49C).Standard error associated with the inhibitor plasma was higher than thatseen in the healthy plasma or hemophilia A pool. This was most likelydue to the difference in titers between the three inhibitor patients(160 BU/mL, 533 BU/mL and 584 BU/mL).

Overall, with the exception of the lag time, ARC19499 and NovoSeven® hadvery comparable effects on thrombin generation in all plasmas tested.

Example 21

This example demonstrates that the TFPI aptamers have biologicalactivity.

In this experiment, the ability of ARC19499 to affect clot formationcompared to that of NovoSeven® was tested using the thromboelastography(TEG®) assay. The TEG® assay measures the mechanical properties of adeveloping clot. In the TEG® assay, a cup containing the blood productand any activators oscillates freely around a pin that is attached to atorsion wire. As a clot develops, newly formed fibrin strands connectthe oscillating cup to the stationary pin and begin to pull on the pin,thus generating force on the torsion wire. This force is converted to asignal by the computer to monitor clot formation, and is displayed as atracing of signal height versus time. From this tracing, one can extracta number of parameters to measure various aspects of clot formation. TheR-value measures the time that it takes for an initial clot to develop.The angle is a measure of the rate at which the clot forms. The maximumamplitude (MA) is a measure of clot strength and stability.

These studies were performed in citrated whole blood from healthyvolunteers. In the first assay, the drugs were tested in untreated wholeblood. In the second assay, the blood was first treated with a sheeppolyclonal antibody against human Factor VIII for three hours at 37° C.prior to drug addition. In both assays, NovoSeven® or ARC19499 wereadded to the blood (antibody-treated or not) at final bloodconcentrations of 0.01, 0.1, 1, 10 or 100 nM. Activation of clottingoccurred upon addition of tissue factor (Innovin) at a final dilution of1:200000 (˜6 fM) and calcium chloride at a final concentration of 11 mM.

In untreated blood, both ARC19499 and NovoSeven® demonstrated adose-dependent, moderate decrease in R-value that appeared to reach aminimum value by 10 nM. (FIG. 50A). The angle and MA values remainedunchanged over the concentrations tested (FIG. 50B-C).

In the blood treated with Factor VIII antibody, both drugs had similareffects on the R-value. The R-value was prolonged in blood treated withantibody compared to untreated blood. As the ARC19499 or NovoSeven®concentration increased, the R-value was restored to the same level itwas in untreated blood (FIG. 51A). Antibody treatment decreased the rateof clot formation in the blood, which is reported as the angle.NovoSeven® had a strong effect on the angle, increasing it linearly from0.1 to 100 nM of NovoSeven®. This increase surpassed the angle achievedwith untreated blood. ARC19499 also increased the angle, but the valueappeared to plateau by 10 nM of aptamer, at a similar level as thatachieved with untreated blood (FIG. 51B). The effect on MA was minimalwith both drugs, primarily because there does not appear to be a largedifference in the MA of whole blood, with or without FVIII antibodytreatment. Both drugs resulted in MA values that fell between thoseachieved with untreated blood and those achieved with antibody-treatedblood (FIG. 51C).

As seen in the CAT assay, ARC19499 and NovoSeven® had very comparableeffects on clot formation in whole blood, whether the blood was lackingFactor VIII or not. The main difference between the two drugs was seenin the effect on the rate of clot formation (angle), with NovoSeven®showing a more linear increase in rate as the concentration increased,but ARC19499 did increase the rate as well.

Example 22

This example demonstrates that the TFPI aptamers have biologicalactivity.

In this experiment, the synergy between ARC19499 and Factor VIII inthrombin generation was tested using the calibrated automatedthrombogram (CAT) assay. These studies were performed in the presence ofa pool of plasma from hemophilia A patients with <1% Factor VIII (FVIII)levels (commercially available from George King Bio-Medical, Inc,Overland Park, Kans.). Increasing concentrations of ARC19499 (from 1 to300 nM) were analyzed in the presence of 0, 1.4, 2.5, 5, 14 and 140%Factor VIII (World Health Organization International Standard). Theresults were compared to the baseline responses for hemophilia A andpooled normal plasmas in the absence of ARC19499.

Assuming the normal and hemophilia A plasmas are otherwise equivalent,the absence of Factor VIII in the hemophilia A plasma caused a marginaldecrease in the baseline lag time for thrombin generation compared tonormal plasma (FIG. 52A), a 3-4 fold decrease in peak thrombinconcentration (FIG. 52B) and a 1.5-1.6 fold decrease in endogenousthrombin potential (ETP) (FIG. 52C) in this experiment. In the absenceof Factor VIII, ARC19499 had little or no effect on the lag time forthrombin generation, but caused a dose-dependent increase in peakthrombin concentration and ETP. The addition of exogenous Factor VIIIcaused incremental changes in all parameters, with the largest effectsobserved on the peak thrombin concentration (FIG. 52B). Reconstitutionwith 140% Factor VIII restored this parameter to a level similar to thatobserved in normal plasma, with smaller improvements observed at 14%Factor VIII and below. Moreover, the incremental increase in peakthrombin caused by each Factor VIII concentration was nearly identicalat all concentrations of ARC19499, suggesting that the effects of thetwo agents on thrombin generation are additive rather than synergistic.With ETP, Factor VIII flattened the ARC19499 dose-response curve with anadditive effect observed only at the lower concentrations of ARC19499(FIG. 52C). Once 10 nM of ARC19499 was reached, additional Factor VIIIdid not appear to have a benefit. FIG. 53A shows the ETP of hemophilia Aplasma with different concentrations of FVIII added (dashed lines).Addition of ARC19499 resulted in a dose-dependent increase in thrombingeneration in hemophilia A plasma and in hemophilia A plasma with 5%FVIII added. ARC19499 mediated a procoagulant effect in hemophilia Aplasma that was similar to 14% FVIII at 1-10 nM aptamer when ETP wasevaluated (FIG. 53A) or 10-30 nM when peak thrombin was evaluated. Whena saturating amount of ARC19499 (300 nM) was added to plasma withdifferent concentrations of FVIII, thrombin generation levels were nearto that observed with normal plasma, indicating that ARC19499 does nothave a severe prothrombotic effect (FIG. 53B). Even with 140% FactorVIII, ETP levels of normal plasma were never reached. By this measure,therefore, the addition of exogenous Factor VIII appeared to obviate theneed for a bypassing agent like ARC19499, rather than facilitate itsaction. Interestingly, ARC19499 appeared to decrease lag time at thehigher concentrations of Factor VIII (FIG. 52A).

The inhibition of TFPI in this case may enable more rapid, FactorVIII-dependent propagation of thrombin generation.

Example 23

This example demonstrates that ARC19499 can improve coagulation in aspatial model of clot formation, in hemophilia plasma activated withimmobilized tissue factor.

The key property of the spatial experimental model is that blood plasmaclotting is activated by a surface covered with immobilized tissuefactor (TF). The fibrin gel then propagates into the bulk of plasma.Clotting takes place in a specially designed chamber (FIG. 54A). Plasmasamples are loaded into the well of the chamber that is subsequentlyplaced in the thermostat. All experiments are performed at 37° C.Clotting is initiated by immersion of an insert with TF immobilized onits end face into the chamber. Clot formation is registered by lightscattering from fibrin gel using a CCD camera (FIG. 54B). The chamber isuniformly illuminated with monochromatic light and images are capturedevery 15 seconds. The acquired series of images is then processed bycomputer and parameters of spatial dynamics of blood clotting arecalculated.

For the purposes of this set of experiments, surfaces were derivatizedwith TF densities in the range of 1-100 pmole/m². The density of TF onthe surface was characterized by the ability to activate Factor X(Enzyme Research Laboratories) in the presence of excess Factor VIIa(Novoseven®; Novo Nordisk) using a chromogenic Factor Xa substrateS-2765 (Chromogenix). The rate of S-2765 cleavage was measured by lightabsorption (405 nm) and compared to a calibration curve prepared using aset of TF standard solutions (American Diagnostica) to calculate TFconcentration.

Each light scattering image was processed by calculating the mean lightscattering intensity (based on pixel intensity) along a perpendiculardrawn to activating surface. The data from each image was depicted as asingle contour line on a plot of light scattering intensity versusdistance from the activating surface (FIG. 55). Clot propagation wasdepicted qualitatively by successive contour lines of increasing lightscattering intensity, determined from images taken at consecutivetimepoints up to 90 minutes (FIG. 55), or quantitatively, by plottingclot size versus time (FIG. 56). The clot size for each image wasdetermined as the coordinate (in micrometers or millimeters) along thecontour line where the scattering intensity is half-maximal. Based onclot size versus time plots, the following parameters were calculated:lag time (delay between contact of plasma with activator and beginningof clot formation), initial velocity of clot growth (α or V_(initial);mean slope of the clot size versus time curve over the first 10 minutesafter the lag time), spatial or stationary velocity of clot growth (β orV_(stationary), mean slope over the next 30 minutes) and clot size after60 minutes of the experiment (an integral parameter of clot formationefficiency). For each experiment, four perpendiculars were drawn fromdifferent points along the activator surface. Profiles of clot sizeversus time were analyzed and four values of each clotting parameterwere obtained and then averaged to obtain means.

This study was conducted primarily using freshly prepared plasma (ratherthan commercial or frozen plasma) from normal donors and hemophilia Apatients. Blood was collected from healthy volunteers and hemophilia Apatients at a 9:1 v/v ratio into a solution containing 3.8% sodiumcitrate plus 0.2 mg/ml CTI (Institute of Protein Research, RussianAcademy of Sciences), then it was processed by centrifugation at 1,500 gfor 15 minutes to obtain platelet-poor plasma. It was additionallycentrifuged at 10,000 g for 5 minutes to obtain platelet-free plasma.Fresh pools of normal plasma were prepared from 3 healthy donors each.At 15 minutes before an experiment, 300 μL of plasma was supplementedwith 18 μL of ARC19499 or recombinant Factor VIIa (alternativelydesignated rVIIa or Novoseven®). In control experiments lacking ARC19499or Factor rVIIa, plasma was supplemented with the same volume ofphosphate buffered saline. Plasma was recalcified by the addition of 60μL 1 M CaCl₂, mixed, and 300 μL of recalcified plasma was placed in theexperimental chamber. The insert with the TF-derivatized surface wasthen placed in the chamber to initiate clotting (FIG. 54A).

The result of a typical spatial clot formation experiment, activated by1 pmole/m² of TF density in normal pooled plasma, without and with 300nM of ARC19499 is shown in FIGS. 55A and B, respectively. The plots showcontours of light scattering as a function of distance from theactivator. The time between two contours is 2.5 minutes and the totaltime of each experiment is 90 minutes. The enhancement in lightscattering at each timepoint in FIG. 55B compared to FIG. 55A indicatesthat addition of ARC19499 improved spatial clot formation. However, theeffects of ARC19499 on clot formation are more clearly seen in a plot ofclot size versus time (FIG. 56) derived from the processed scatteringdata, where improvements in lag time, V_(initial) (α) and clot size at60 minutes are observable.

Clot formation parameters were plotted as a function of TF surfacedensity in FIG. 57. Vertical error bars indicate standard deviations(SD) for clot parameters while “n” is the number of experimentsperformed at a specific TF density. Horizontal bars are SD fordeterminations of TF density (n=2 for each activator series). FIG. 57Ashows averaged lag time dependence on activator TF density. Themagnitude of TFPI inhibition by ARC19499 depended on TF density andbecame more significant as the density decreased (up to 2.5-foldshortening of the lag time at TF densities of 1-3 pmole/m²). FIG. 57Bshows the averaged initial clot growth velocity dependence on activatorTF density. Again, the effect of ARC19499 was significant only at low TFdensities (1-3 pmole/m²) where a ˜1.8-fold increase of the initialvelocity was observed. FIG. 57C illustrates the stationary clot growthvelocity dependence on activator TF density; ARC19499 had little effecton clot propagation velocity throughout the entire range of activators.Finally, FIG. 57D shows the averaged clot size after 60 minutes.Inhibition of TFPI affected clot size at densities of 1-4 pmole/m² TF;ARC19499 effects became insignificant as the TF density increased. Basedon these data, two TF densities were chosen for further studies ofARC19499: low, 1-2 pmole/m², and medium, 10-20 pmole/m². Several lots ofactivators were prepared for each of these TF densities; the mean valuesfor the low and medium density activators were 2.0±0.68 (n=22 lots) and20.6±8.90 pmole/m² (n=5 lots), respectively.

The influence of different ARC19499 concentrations (from 0 to 1000 nM)on spatial clotting in normal pooled plasma was evaluated to examine thedose-dependence of ARC19499 effects. FIG. 58 shows means and standarderrors of the mean (SEM) for experiments with different normal plasmapools (n=4) and low surface TF densities. Lag time (FIG. 58A) decreasedwith increasing ARC19499 concentration up to 30 nM, and then stabilized.Initial velocity (FIG. 58B) increased by ˜30% with increasing ARC19499concentration, while stationary velocity (FIG. 58C) was notsignificantly affected throughout the entire range of concentrations.There was a detectable increase in clot size at 60 minutes (FIG. 58D)with increasing ARC19499 concentration. For all affected parameters,maximal effects of ARC19499 were clearly achieved by 300 nM, and theconcentration of half-maximal effect was <10 nM. FIG. 59 shows means(±SEM) of clotting parameters for 0 and 300 nM of ARC19499 at low TFdensity combining the raw data from FIGS. 57 and 58 (n=6). To calculatethe statistical significance of the ARC19499 effect, the differencebetween each parameter value, with and without ARC19499, was calculatedfor each experiment, and the distribution of these differences wascompared with zero using the t-test. Asterisks indicate statisticalsignificance (P<0.05), that the difference between values±ARC19499 wasdifferent from zero. The effects on all four parameters werestatistically significant, although the effects on lag time and clotsize were the largest.

FIG. 60 shows mean parameters (±SEM) for experiments with differentnormal plasma pools (n=3) and medium surface TF densities, plotted as afunction of ARC19499 concentration. The effects of ARC19499 on clottingwere less substantial in this experiment. FIG. 61 shows the statisticalanalysis comparing 0 and 300 nM ARC19499 for all four clottingparameters. Although some of the differences appear statisticallysignificant (indicated by asterisk), the effects of ARC19499 on clottingin normal plasma activated by medium TF density were very small.

Typical spatial clot formation activated by low density TF in hemophiliaA plasma is shown in FIG. 62. The plots show profiles of lightscattering as a function of distance from the activator for hemophilia Aplasma alone (FIG. 62A) and hemophilia A plasma containing 100 nMARC19499 (FIG. 62B) or 100 nM rVIIa (FIG. 62C). Example light scatteringimages from which this data was derived are shown in FIG. 63, and a plotof clot size versus time derived from the processed data is shown inFIG. 64, with a normal plasma profile included for comparison. Based onthese data, ARC19499 improved spatial clot formation by shortening lagtime and increasing clot size. As shown in FIG. 64, 100 nM ARC19499partially normalized clot formation, facilitating clot propagation fromthe activating surface. In contrast, 100 nM rVIIa stimulated potentTF-independent clotting. Rather than stimulating normalization ofspatial clot propagation from the activating surface, rVIIa at thisconcentration induced clotting throughout the reaction chamber.

Further experiments characterized the concentration-dependent effects ofARC19499 in various hemophilia patient plasmas. The demographics of thepatient pool from which samples were drawn are shown in the table inFIG. 65. All of the patients had severe (<1%) or moderate (1-5%) FVIIIdeficiencies. FIGS. 66, 67 and 68 show the effects of ARC19499 and rVIIaon spatial clot formation activated by low density TF in plasmas ofpatients #1, #2 and #3, respectively. The error bars henceforth indicateSEM for n=4 regions along the propagating fibrin clot front within asingle experiment. Panels A and B of these figures show the lag timedependence on ARC19499 and rVIIa concentrations, respectively. The lagtime decreased 2-fold with increasing concentrations of ARC19499 from 0up to 30 nM, with no significant further change in lag time at higherARC19499 concentrations. Panels C and D of the same figures show theinitial velocity dependence on ARC19499 and rVIIa concentrations,respectively. The initial velocity increased 2-fold with increasingconcentrations of ARC19499 from 0 up to 30 nM, with no significantfurther change at higher ARC19499 concentrations. FIG. 69 showsstationary clot growth velocity dependence on ARC19499 and rVIIaconcentrations for all 3 patients in one graph. ARC19499 had no effecton stationary velocity through the whole investigated range ofconcentrations, while addition of rVIIa led to a strong increase of thisparameter. Finally, FIG. 70 shows the dependence of clot size at 60minutes on ARC19499 (FIG. 70A) and rVIIa (FIG. 70B). The clot size at 60minutes increased 1.5-2 fold with increasing concentrations of ARC19499from 0 up to 30 nM, with no significant further change at higherARC19499 concentrations. A statistical analysis of the low TF densitydata, comparing each parameter for 0 and 300 nM ARC19499, is shown inFIG. 71. ARC19499 had significant effects on lag time (FIG. 71A),initial velocity (FIG. 71B) and clot size at 60 minutes (FIG. 71D), butno effect on stationary velocity (FIG. 71C).

FIGS. 72, 73 and 74 show the effects of ARC19499 and rVIIa on spatialclot formation activated by medium density TF in plasmas of patients #4,#5 and #6, respectively. ARC19499 had little effect on clottingparameters for the medium density activator through the entire range ofconcentrations tested. A statistical analysis of the medium TF densitydata comparing each parameter for 0 and 300 nM ARC19499 is shown in FIG.75. ARC19499 had no significant effect on any of the four clottingparameters under these conditions.

To estimate the extent of clotting normalization by ARC19499 underconditions of low TF density, FIG. 76 shows the mean parameters ofclotting for hemophilia A and hemophilia A with 300 nM of ARC19499 incomparison to normal plasma. ARC19499 shortened the lag time below thenormal level and normalized the initial velocity, but had no effect onstationary velocity. ARC19499 increased the clot size at 60 minutesapproximately 2-fold from 30% up to 60% of the normal value. In order tocheck whether ARC19499 effects differ in normal and hemophilia A plasma,the ratios with and without 300 nM of ARC19499 were plotted for all fourclotting parameters[ratio=(Parameter)_(+ARC19499)/(Parameter)_(−ARC19499)] (FIG. 77). Inboth hemophilia A and normal plasmas, the lag time ratio was ˜0.5,indicating that the addition of 300 nM ARC19499 decreased the lag timeby about half in each. The ratios for V_(stationary) were also similarbetween plasmas. However, larger ratios were observed for V_(initial)and clot time at 60 minutes in hemophilia A plasma compared to normalplasma, suggesting that the maximal effect of ARC19499 in hemophilia Aplasma slightly exceeded that in normal plasma.

To determine an IC₅₀ for ARC19499 in hemophilia A plasma, we plottedmean lag time and clot size as a function of ARC19499 concentration (upto 10 nM) for low TF density (FIG. 78). The half-maximal effect,calculated by curve-fitting, was ˜0.7 nM for both parameters.

FIG. 79 compares clotting parameters for hemophilia A plasma alone with300 nM ARC19499 or with 30 nM rVIIa. FIG. 79A-D show lag time, initialclot growth velocity, stationary velocity and clot size after 60minutes, respectively. In contrast to rVIIa, ARC19499 increased clotsize primarily by shortening the lag time and increasing initialvelocity; it had no effect on spatial propagation stage(V_(stationary)).

Experiments at low TF density in TFPI-depleted plasma were alsoperformed to gain insight into the mechanism of action of ARC19499 andinto the regulation of spatial clotting by TFPI. LyophilizedTFPI-depleted plasma was purchased from American Diagnostica,resuspended in deionized water, and CTI added to 0.2 mg/mL. RecombinantTFPI (rTFPI; R&D Systems) was added into plasma at the concentration of0 or 10 nM, with or without ARC19499 (0 or 300 nM), for measurement ofspatial clot formation in the presence of low TF surface density. Theaddition of rTFPI significantly increased the lag time (FIG. 80) in theabsence of ARC19499, but had no effect in the presence of 300 nMARC19499, suggesting that it was completely inhibited. ARC19499 had noeffect on clotting in TFPI-depleted plasma in the absence ofsupplementary rTFPI, indicating that its effects are TFPI-specific.Neither rTFPI or ARC19499 had any effect on initial velocity in thisexperiment.

In conclusion, ARC19499 significantly improved clotting in normal andhemophilia A plasma in the spatially heterogeneous system at low TFdensity (1-3 pmole/m²). The lag time was shortened, and initial velocityof spatial propagation and clot size at 60 minutes were increased byARC19499 up to 2-fold, with little effect on spatial propagationvelocity far from the activator. In hemophilia A plasma, this resultedin complete normalization of the lag time and initial velocityparameters, while clot size at 60 minutes was partially normalized(increases from 30% to 60% of normal upon addition of ARC19499). Withincreases in TF density, the effects of the aptamer became smaller andthere was almost no effect at TF>20 pmole/m². The action of ARC19499 onclotting in this experiment at low TF density was TFPI-specific, sinceARC19499 had no effect on clotting in TFPI-deficient plasma.

Example 24

This example demonstrates that ARC19499 can improve clotting in wholeblood and cell-free (plasma) clot-time assays, in samples collected fromhemophilia A and hemophilia B patients.

Blood samples (20 mL) were collected from 12 subjects, including sevensevere hemophilia A (subjects #1, 3, 5, 8, 10, 11 and 12) subjects, twosevere hemophilia B (#4 and 9) subjects and three healthy controls (#2,6 and 7). Blood was collected into 0.5 mM EDTA and 0.1 mg/mL corntrypsin inhibitor (CTI; Haematologic Technologies Inc.). Approximatelyhalf of each sample was used for whole blood assays, while the otherhalf was centrifuged to prepare platelet poor plasma (PPP).

The TF-activated clotting time (TF-ACT) is a whole blood assay performedusing the Hemochron® Response Whole Blood Coagulation System(International Technidyne Corp.), a commonly used system for measuringpatient responses to unfractionated heparin and protamine. Standard ACTsmeasured by this instrument use tubes containing an activator of the“contact” or “intrinsic” pathway of coagulation (e.g., celite orkaolin). However, for TF-ACTs, the tubes designed for measuring standardACTs were rinsed of contact-activating reagent. In its place was added12 μL of 1 M CaCl₂, a desired amount of ARC19499, and 2 μL of 5 nMrelipidated, recombinant TF (Haematologic Technologies). Upon theaddition of 2 mL whole blood to this mixture, the clot time was measuredon the Hemochron® Response instrument as for a standard ACT. The resultsare shown in tabular format in FIG. 81. An average baseline TF-ACT of335±22 seconds was observed in normal subjects. Moderate decreases inTF-ACT (up to 75 seconds), indicative of a procoagulant effect, wereobserved in these individuals for ARC19499 concentrations ranging from44 to 700 nM. The two hemophilia B subjects showed baseline TF-ACTs of528 and 580 seconds. The TF-ACT decreased by 160-205 seconds at 88 nMARC19499 in these two individuals, then increased moderately at higherARC19499 concentrations (up to 350 nM). A relatively broad range ofbaseline TF-ACT values was observed in the hemophilia A group, with anaverage value of 578±140 seconds. Substantial ARC19499-dependentshortening of the TF-ACT was observed in 6 of 7 of these individuals,and in two of these (subjects #1 and 11) values in the normal range orbelow were observed. Only a moderate decrease of up to 47 seconds wasobserved in subject #12, but this subject also displayed the shortestbaseline TF-ACT (328 seconds) of the group. These data suggest that TFPIsuppression by ARC19499 was able to improve clotting activity in asimple, whole blood clotting assay.

Dilute prothrombin time (dPT) assays were performed on PPP prepared fromthe same blood samples as described for the TF-ACT assays. The standardprothrombin time (PT) is performed by adding thromboplastin, consistingof tissue factor (˜1 nM), calcium chloride and phospholipids, to plasmato evaluate the integrity of the “tissue factor” or “extrinsic” pathwayof coagulation. The clot time in a normal plasma sample measured usingthe standard PT protocol is typically ˜11 seconds. The PT is commonlyused for measuring patient responses to warfarin, and is largelyinsensitive to deficiencies in contact pathway factors like FVIII andFIX. In contrast to the standard PT, the dPT uses a very low TFconcentration and clot times measured by this assay are sensitive tofactors in both the TF and contact pathways. In this particularexperiment, thromboplastin reagent (Innovin; Dade-Behring) was dilutedin tris-buffered saline (20 mM tris, pH 7.5, 150 mM NaCl) to reach aconcentration of 0.3 pM TF. The dPT was performed by mixing 120 μL ofPPP with 60 μL of the dilute TF solution and incubating at 37° C. for 3minutes before adding 60 μL of 25 mM CaCl₂ to the plasma/TF mixture.Clotting time was recorded on an ACL-8000 coagulometer (fromInstrumentation Laboratory, Bedford, Mass.) and the data for allsubjects is shown in tabular format in FIG. 82. Baseline clot times inPPP samples from all normal and hemophilia B subjects, and 6 of 7hemophilia A subjects were >360 seconds, which was the pre-set, maximummeasurable clot time on the coagulometer. One hemophilia A subject (#10)displayed a baseline dPT of 169 seconds. Increasing concentrations ofARC19499 added to the PPP typically resulted in decreased dPT clottimes. In PPP from normal subjects, 2 nM ARC19499 was sufficient tosignificantly lower the clot time (avg=278±15 seconds) relative tobaseline, but had no apparent effect in plasma from hemophilia A or Bsubjects. However, 8 nM ARC19499 lowered the clot time in PPP fromnearly all subjects. Exceptions were observed in PPP from subject #10,where a low baseline dPT was observed, and #12, who appearedunresponsive to ARC19499. Excluding these two individuals, the averageclot time in the hemophilia A group for 8 nM ARC19499 was 188±8 seconds.The average clot times for the normal and hemophilia B groups under thesame conditions were 204±37 seconds and 226±22 seconds, respectively.Higher ARC19499 concentrations caused only moderate, further decreasesin clot times. Average clot times at 500 nM ARC19499 were 179±6 seconds,200±21 seconds and 161±3 seconds for the normal, hemophilia A (excluding#10 and #12) and hemophilia B groups, respectively. These data indicatethat TFPI suppression by ARC19499 was able to improve clotting activityin a simple, plasma-based clotting assay.

Example 25

This example demonstrates that ARC19499 can improve clotting in wholeblood samples from hemophilia A and hemophilia B patients, as measuredby rotation thromboelastometry (ROTEM).

Blood samples were collected from 39 healthy volunteers (27 male and 12female) and 40 hemophilia patients (all male). Of the 40 hemophiliapatients, 3 hemophilia B (HB) patients and 28 hemophilia A (HA) patientswere diagnosed as severe (baseline factor activity <1%), one HA and oneHB patient suffered from moderately severe hemophilia (baseline factoractivity 1-5%), four HA and two HB patients had mild hemophilia(baseline factor activity>5%). Using a 21-gauge butterfly needle, bloodsamples were drawn into plastic Vacuette tubes (Greiner Bio-One)containing 3.8% sodium citrate at a volume ratio of 1:9.

Coagulation was analyzed by ROTEM (Pentapharm GmbH), which is based onthe original thromboelastography system (TEG™). In a typical ROTEMexperiment, blood is incubated at 37° C. in a heated cup. As fibrinforms between the cup and the pin, the impedance of the rotation of thepin is detected and a trace is generated, indicating clot formation overtime. The following parameters may be analyzed from the ROTEM trace: theclotting time (CT), the clot formation time (CFT), the maximum clotfirmness (MCF) and the alpha angle (alpha). The clotting time (CT)characterizes the period from analysis start until initiation of theclot. The clot formation time (CFT) describes the subsequent perioduntil an amplitude of 20 mm is reached. The alpha angle is given by theangle between the center line and a tangent to the curve through the 2mm amplitude point. Both the CFT and the alpha angle denote the speed ofclot development. The MCF is calculated from the maximum amplitude ofthe ROTEM trace and describes clot stability and strength; the MCF islargely dependent on fibrinogen and platelet function.

In this set of experiments, 300 μL of ARC19499-spiked whole blood (0,0.2, 0.6, 2, 6, 20, 60, 200 or 600 nM ARC19499) was transferred intopre-warmed plastic cups. Blood samples were recalcified using 20 μL 0.2M CaCl₂ and coagulation was activated by ˜33 fM tissue factor (TF)(Innovin, Dade Behring, diluted 1:200,000). All analyses were performedat 37° C. Measurement was performed either without corn trypsininhibitor (CTI) or with the addition of 100 μg/mL of CTI (HaematologicTechnologies Inc). Comparisons between different concentrations ofARC19499 in any of the measured parameters were calculated with theWilcoxon signed rank test with a Bonferroni Correction. To comparehealthy controls to patients, a Mann-Whitney U-test was used. To analyzethe correlation of hemostatic parameters to Factor VIII (FVIII)activity, the Spearman's rank correlation coefficient was used. Ap-value smaller or equal to 0.05 was considered statisticallysignificant.

The baseline whole blood clotting profile of hemophilia patients wascharacterized by a prolonged initiation phase (as shown by a prolongedCT) and a diminished propagation phase of whole blood clotting(prolonged CFT and a lower alpha angle) compared to healthy controls(p<0.01 for all) in the absence of CTI (FIG. 83).

Concentrations of ARC19499≧2nM enhanced whole blood coagulationsignificantly in both hemophilia patients and healthy controls (p<0.01).The maximum hemostatic effect of ARC19499 was achieved withconcentrations≧60 nM. In hemophilia blood, ARC19499 decreased the CFTand increased the alpha angle to values equal to those of healthycontrols (p>0.4). The clot time was substantially improved by ARC19499,but not fully normalized. The MCF, though not significantly differentbetween controls and patients, was significantly augmented by ARC19499(p<0.05 for 0 nM ARC19499 compared to ≧2 nM in hemophilia patients and200 nM in healthy controls).

A comparison between FVIII coagulant activity (FVIII:C) activity levelsin the hemophilia A patient samples and baseline CT indicated asignificant correlation (p<0.01). Therefore, the hemophilia A patient CTdata were stratified into three groups (<1% FVIII:C, 1-5% FVIII:C,and >5% FVIII:C) and replotted next to the healthy control CT data (FIG.84). As previously indicated, concentrations>2 nM ARC19499 significantlyshortened the clotting time (p<0.01). The CT values for hemophiliapatients and healthy controls remained significantly different (p<0.05),but ARC19499 shortened the CT of hemophilia patients by a maximum up to38%, and those of healthy controls by up to 19%, compared to baselinevalues. ARC19499 had the largest effect on the CT in patients withmeasured FVIII:C<1%. Although the CT of patients with FVIII:C<1% was notentirely normalized, ARC19499 shortened the CT to the range of healthycontrols and to values equal those of patients with an FVIII:C>5%.

Additional ROTEM analyses were performed in blood containing CTI usingsamples from 28 hemophilia patients and 11 healthy male controls. Again,the coagulation profiles were significantly different for patients andhealthy controls (p<0.01) for all parameters except the MCF (FIG. 85).Similar to the measurements in the absence of CTI, addition of ARC19499to blood containing CTI significantly shortened the CT and CFT, andraised the alpha angle and MCF (concentrations≧20 nM, p≦0.01). In bloodcontaining CTI, the pro-haemostatic effect was more pronounced than inblood without CTI. Upon addition of 200 nM of ARC19499 to bloodcontaining CTI, the CT was not only shortened significantly (p<0.01) butwas also no longer different from the baseline CT of healthy controls(p=0.06). ARC19499 also normalized the CFT and the alpha angle in CTIwhole blood, as previously observed in whole blood lacking CTI. ROTEMparameters of hemophilic blood spiked with ≧60 nM of ARC19499 were equalto baseline values of healthy controls (p>0.1).

One patient with acquired hemophilia A was recruited. This patientshowed a FVIII:C activity of 7%, 8.5 BU/mL FVIII inhibitor and anelevated aPTT of 63 seconds. This patient had received two infusions ofFVIII bypassing activity (FEIBA), the most recent one within 8 hours ofvenipuncture. As a consequence, the CT and CFT of this patient werealready normal (patient values: CT=496 seconds, CFT=213 seconds; healthycontrol mean values: CT=607 seconds, CFT=251 seconds). ARC19499shortened the CT and CFT further, and even more than in controls andhereditary hemophilia patients (CT: 47% vs. 19% and 30%, CFT: 45% vs.38% and 22%). ARC19499 also increased the alpha angle, as shown in FIG.86.

Since patients with acquired hemophilia are extremely rare, the ROTEMexperiment was repeated on normal blood that had been treated with aneutralizing antibody to FVIII. Blood from healthy controls waspre-incubated with a sheep antihuman FVIII polyclonal antibody (specificactivity 2300 BU/mg; Haematologic Technologies Inc). FIG. 87 shows theCT (left panel) and the CFT (right panel) in the same controls; on theleft side of each graph, values after inhibition by the FVIII antibodyare depicted. The addition of 60 nM ARC19499 normalized both the CT andthe CFT in antibody-treated blood.

These data show that ARC19499 had a procoagulant effect on clotting inblood samples from healthy controls and hemophilia patients, as measuredby ROTEM. Additionally, ARC19499 was able to normalize ROTEM clottingparameters in blood from hemophilia patients.

Example 26

This example demonstrates that ARC19499 can improve thrombin generationin plasma samples from hemophilia A and hemophilia B patients, asmeasured by calibrated automated thrombography (CAT).

Blood samples were collected from 39 healthy volunteers (27 male and 12female) and 40 hemophilia patients (all male). Of the 40 hemophiliapatients, 3 hemophilia B (HB) patients and 28 hemophilia A (HA) patientswere diagnosed as severe (baseline factor activity <1%), one HA and oneHB patient suffered from moderately severe hemophilia (baseline factoractivity 1-5%), and four HA and two HB patients had mild hemophilia(baseline factor activity >5%). Using a 21-gauge butterfly needle, bloodsamples were drawn into plastic Vacuette tubes (Greiner Bio-One)containing 3.8% sodium citrate at a volume ratio of 1:9. Platelet poorplasma (PPP) was prepared from these samples by two room temperaturecentrifugation steps, with the first spin at 1700×g for 10 minutesfollowed by a second spin at 18,000×g for 15 minutes. PPP containing 100μg/mL corn trypsin inhibitor (CTI; Haematologic Technologies Inc.) wasspiked with different ARC19499 concentrations (0, 0.2, 0.6, 2, 6, 20,60, 200 or 600 nM ARC19499) for use in the assay.

CAT assays were performed by adding 80 μL of PPP to 204 of a mixture oftissue factor (TF) and phospholipids (PPP-Reagent Low, ThrombinoscopeBV) in a 96 well microtiter plate. The final concentrations of TF andphospholipids were 1 pM and 4 μM, respectively. The reaction was startedby adding 20 μL of fluorogenic substrate (FluCa Kit, Thrombinoscope BV)and fluorescence was detected using a Fluoroskan Ascent fluorometer(Thermo Fisher Scientific). Analysis by Thrombinoscope software resultedin thrombin generation curves with thrombin (nM) on the y-axis and time(minutes) on the x-axis. The software determined values for a number ofparameters including: lag time (minutes; time till onset of initialthrombin generation); endogenous thrombin potential (ETP; nM; area underthe thrombin generation curve); peak thrombin (nM; highest amount ofthrombin generated at any one point of the assay); time to peak(minutes; time reach peak thrombin concentration); and start tail(minutes; point in time when the end thrombin generation is reached).Comparisons between different concentrations of ARC19499 in any of themeasured parameters were calculated with the Wilcoxon signed rank testwith a Bonferroni Correction. To compare healthy controls to patients, aMann-Whitney U-test was used. A p-value smaller or equal to 0.05 wasconsidered statistically significant.

Examples of CAT data are shown in FIG. 88 for a representativehemophilia A patient (left panel) and a healthy control (right panel),comparing thrombin generation in the presence and absence of 200 nM ofARC19499. The addition of ARC19499 normalized the thrombin generationcurve in the hemophilia A patient sample and augmented thrombingeneration in the healthy control sample.

Average CAT parameters for all hemophilia patients and healthy malecontrols are shown in FIG. 89. At baseline, the hemophilia patient groupdisplayed a prolonged time to peak, a lower peak thrombin generation anda severely compromised ETP. In the absence of ARC19499, the followingCAT parameters were significantly different between healthy malecontrols and hemophilia patients: time to peak, peak thrombingeneration, ETP and start tail (p<0.001). There was no significantdifference in the lag time between healthy controls and hemophiliapatients (FIG. 90). The addition of ARC19499 significantly enhanced allof the parameters assessed by the CAT assay (FIGS. 89 and 90).Concentrations above 60 nM of ARC19499 normalized CAT parameters inhemophilia patients. There was no longer a significant differencebetween baseline values of healthy controls and values measured withaddition of ≧60 nM ARC19499 in hemophilia patients in the following CATparameters: start tail, time to peak and ETP (p>0.05) (FIG. 89). Peakthrombin remained significantly different between patients and controls,but 600 nM of ARC19499 augmented peak thrombin by 185% in hemophiliapatients, from 47 nM at baseline to 135 nM, thus reaching the normalrange of healthy controls (healthy controls mean peak thrombin 167 nM,minimum 100 nM, maximum 297 nM). ARC19499 shortened the lag timesignificantly in both groups (p<0.001 for concentrations above 0.6 nM)but differences between the groups continued to be not significant (FIG.90).

FIG. 91 shows peak thrombin data stratified into three groups (<1% FVIIIcoagulant activity (FVIII:C), 1-5% FVIII:C, and >5% FVIII:C) andreplotted next to the healthy control peak thrombin data. Increasingconcentrations of ARC19499 augmented peak thrombin generation in allfour groups. ARC19499 did not completely normalize peak thrombin in the<1% FVIII:C plasma relative to the average healthy control value atbaseline, but values at 60 nM ARC19499 reached higher than the baselineof patients with >5% FVIII:C concentration and into the range of valuesobserved for healthy controls (hatched region in FIG. 91).

CAT assays were also performed on ARC19499-spiked samples from a patientwith acquired hemophilia. This patient showed a FVIII:C activity of 7%,8.5 BU/mL FVIII inhibitor and an elevated aPTT of 63 seconds. Thispatient had received two infusions of FVIII bypassing activity (FEIBA),the most recent one within 8 hours of venipuncture. As a consequence,ROTEM parameters of clotting in this patient were already normal(patient values: CT=496 seconds, CFT=213 seconds; healthy control meanvalues: CT=607 seconds, CFT=251 seconds). Thrombin generation curvesmeasured in the presence of 0, 2, 20 and 200 nM ARC19499 are shown inFIG. 92. Although this patient displayed normal ROTEM values, baselineCAT values were severely compromised. As for patients with inheritedhemophilia, increasing concentrations of ARC19499 normalized thrombingeneration in samples from this patient. ARC19499 had the largestinfluence on the peak thrombin and the ETP of the patient, both of whichincreased more than 2.5 fold. Peak thrombin was normalized (peakthrombin: baseline 54 nM-max 165 nM; mean baseline value of healthycontrols: 164 nM). Moreover ARC19499 (200 nM) increased the ETP tovalues above those of healthy controls (ETP: baseline 973 nM-max 2577nM; mean baseline healthy controls: ETP 1322 nM).

The CAT experiment was repeated on normal plasma that had been treatedwith a neutralizing antibody to FVIII. PPP from healthy controls waspre-incubated with a sheep antihuman FVIII polyclonal antibody (specificactivity 2300 BU/mg; Haematologic Technologies Inc). FIG. 93 shows theETP (left panel) and the peak thrombin (right panel) in the samecontrols; on the left side of each graph, values after inhibition by theFVIII antibody are depicted. The addition of 60 nM ARC19499 normalizedboth the ETP and the peak thrombin in antibody-treated plasma.

These data show that ARC19499 had a procoagulant effect on thrombingeneration in blood samples from healthy controls and hemophiliapatients, as measured by CAT. Additionally, ARC19499 was able tonormalize CAT parameters in plasma from hemophilia patients.

Example 27

This example demonstrates that ARC19499 can improve thrombin generationtimes (TGT) in plasma samples from severe, moderate and mild hemophiliaA patients, and severe hemophilia B patients, as measured by calibratedautomated thrombogram (CAT).

Blood was collected into 3.2 mL Vacuette tubes containing 3.2% sodiumcitrate and 250 μL 1.3 mg/mL corn trypsin inhibitor (CTI); the final CTIconcentration was 100 μg/mL. Samples were collected from patients withsevere (<1% FVIII; n=10), moderate (1-5% FVIII; n=7) and mild (5-40%FVIII; n=5) hemophilia A, patients with severe hemophilia B (<1% FIX,n=5) and healthy volunteers (n=10). To prepare platelet poor plasma(PPP) for CAT assays, tubes were centrifuged at 2500×g for 15 minutes,the supernatant transferred to fresh Eppendorf tubes, then centrifugedagain at 11000×g for 5 minutes. Plasma was either used immediately orfrozen at −80° C. for later use. To analyze the effects of ARC19499 onthrombin generation, ARC19499 was added to plasma at concentrations of10, 100, 300, 1000, 3000 or 10,000 ng/mL (0.9, 9.0, 27.1, 90.3, 271 or903 nM).

Thrombin generation time (TGT) assays were performed by calibratedautomated thrombography (CAT) on a Thrombinoscope instrument(Thrombinoscope, Maastricht, The Netherlands) consisting of a ThermoScientific Fluoroskan Ascent Microplate Fluorometer (serial no.374-90031C) programmed with Thrombinoscope software version 2.6. 80 μLplasma was mixed with 20 μL relipidated recombinant tissue factor (TF;final concentration 1 pM) and the assay was initiated by the addition of20 μL FluCa substrate. Analysis of the data by Thrombinoscope softwareresulted in thrombin generation curves with thrombin (nM) on the y-axisand time (minutes) on the x-axis. The software determined values for anumber of parameters including: lag time (minutes; time till onset ofinitial thrombin generation), endogenous thrombin potential (ETP; nM;area under the thrombin generation curve), peak thrombin (nM; highestamount of thrombin generated at any one point of the assay) and time topeak (minutes; time reach peak thrombin concentration). All measurementswere performed in duplicate. Differences in means of each group weretested using a student's t-test or ANOVA, as appropriate.

FIGS. 94-97 show representative CAT data from a healthy volunteer (HV),a patient with severe hemophilia A (SHA), a patient with moderatehemophilia A (MoHA) and a patient with mild hemophilia A (MiHA). Mediandata for healthy volunteers, all three hemophilia A patient groups, andthe severe hemophilia B patient group are shown in FIG. 98 forexperiments performed in freshly processed plasma. Baseline ETP and peakthrombin parameters were decreased in all hemophilia patient groupscompared to healthy controls, and the time to peak was increased. Theseverity of FVIII deficiency had no effect on observed thrombingeneration, as baseline parameter values were essentiallyindistinguishable between the 3 hemophilia A patient groups. FVIIIdeficiency had little effect on the baseline lag time compared tohealthy controls, but FIX deficiency resulted in ˜2-fold prolongation ofthe lag time at baseline. Freezing the plasma had little effect onthrombin generation as similar CAT parameter values were observed inplasma samples that had undergone freeze-thaw (FIG. 99).

As shown in the individual (FIGS. 95-97) and median data plots (FIGS. 98and 99), the addition of ARC19499 improved thrombin generation inhemophilia plasma. The ETP increased with increasing ARC19499concentrations up to 10,000 ng/mL (903 nM) reaching a normal level inall hemophilia groups (FIGS. 98 and 99). A trend toward improvement wasalso observed in the peak thrombin. Although normalization of thisparameter was not achieved, 3-5-fold improvements were observed in allpatient groups. ARC19499 had little effect on the lag time in any of thehemophilia A patient groups, but it did slightly improve the lag time inplasma from hemophilia B. A slight improvement in time to peak was alsoobserved in all of the patient groups. In healthy volunteer plasma, theaddition of ARC19499 had little effect on any of the CAT parameters.

Median CAT data with interquartile ranges are presented for fresh andfrozen plasma in the following tables: Table 3, severe hemophilia A(SHA); Table 4, moderate hemophilia A (MoHA); Table 5, mild hemophilia A(MiHA); Table 6, severe hemophilia B (SHB); and Table 7, normal. Takentogether, the data show that ARC19499 improved thrombin generation inhemophilia A patient plasma of all severity levels, and in severehemophilia B plasma.

TABLE 3 TGT on Citrated PPP with CTI - SHA (n = 10) ETP[nM/min] Peak[nM]ARC19499 Fresh Frozen Fresh Frozen ng/mL Median IQR Median IQR MedianIQR Median IQR 10000 1347 1260-1538 1587 1244-1763 88  63-119 107 86-1393000 1233 1117-1425 1569 1094-1716 76  56-103 103 77-132 1000 11711019-1335 1546 1019-1711 71 50-95 90 67-125 300 1078  825-1206 1522 916-1657 60 44-77 83 58-117 100 896  741-1137 1206  726-1469 46 36-7658 42-94  10 638 564-978 705  516-1169 34 23-48 31 19-62  0 489 292-711685 247-847 21 13-29 26 10-35  Lag time [min] TT peak[min] ARC19499Fresh Frozen Fresh Frozen ng/mL Median IQR Median IQR Median IQR MedianIQR 10000 3.3 3.2-4.2 3.5 2.9-3.8 10.1 8.6-12.1 10.6 9.1-13.0 3000 3.33.2-3.7 3.5 2.6-4.1 10.1 9.6-11.7 11.5 9.3-13.0 1000 3.3 3.24-3.8  3.63.0-3.8 11 9.6-11.7 9.9 9.4-11.4 300 3.4 3.3-3.8 3.7 2.9-4.0 10.19.5-12.5 11.3 9.9-11.9 100 3.3 2.5-3.7 3.6 3.0-4.2 10.5 9.3-12.3 11.910.8-12.7  10 3.3 3.0-3.5 3.3 2.5-4  10.6 9.3-13.6 11.8 9.2-14.1 0 3.63.4-4.3 4.1 3.6-5.7 17.8 16.2-22.9  22.5 15.5-24.8 

TABLE 4 TGT on Citrated PPP with CTI - MoHA (n = 7) ETP[nM/min] Peak[nM]ARC19499 Fresh Frozen Fresh Frozen ng/mL Median IQR Median IQR MedianIQR Median IQR 10000 1548 1468-1672 1706 1406-2062 102.71  86-134 9787-133 3000 1498 1331-1538 1637 1188-2054 99.39  68-130 94 88-130 10001366 1235-1603 1589 1212-2058 88.92  58-134 86 80-126 300 1314.5 965-1781 1349 1099-1762 81.25 57-90 81 68-108 100 919  835-1792 1085 939-1763 58.77 48-85 74 51-100 10 806  576-1102 796  740-1308 49.1927-60 47 34-87  0 517.9 384-872 764  392-1105 22.62 16-42 41 16-64  Lagtime [min] TT peak[min] ARC19499 Fresh Frozen Fresh Frozen ng/ml MedianIQR Median IQR Median IQR Median IQR 10000 4 3.5-4.8 5.1 4.0-5.6 12.710.5-13.6 12.8 11.3-14.7 3000 4.2 4.0-4.8 5 3.3-5.5 12.5 10.3-13.4 12.611.3-13.1 1000 4.2 4.8-5.8 5.8 4.6-6.0 12.3 10.5-12.6 12.4 11.3-14.1 3004.1 3.6-4.3 3.8   3-5.33 11.2 10.3-13.3 11.0  9.5-12.2 100 4.2 3.5-4.74.8 3.3-5.5 11.0 10.8-13.3 11.5  10-14.1 10 4.5 3.6-4.6 4.6 3.3-6.0 13.710.5-14.6 12.2 11.3-14.6 0 4.8 4-5.8.9 5.0 4.2-7  16.4  15-2.3 15.513.5-16.8

TABLE 5 TGT on Citrated PPP with CTI - Mild HA PPP (n = 5) ETP[nM/min]Peak[nM] ARC19499 Fresh Frozen Fresh Frozen ng/ml Median IQR Median IQRMedian IQR Median IQR 10000 1366 1081-1472  1361 1271-1708 93  75-138 98 87-112 3000 1187 913-1412 1273 1226-1520 87  65-116 81 72-97 1000 1097871-1398 1245 1156-1330 82  62-102 77 72-88 300 1069 804-1317 11361026-1201 78 54-80 69 59-76 100 986 643-1149 1045  753-1145 62 37-77 6140-67 10 709 421-1000 692  551-1068 42 19-52 39 25-58 0 532 337-823  493372-800 28 14-37 25 16-41 Lag time [min] TT peak[min] ARC19499 FreshFrozen Fresh Frozen ng/ml Median IQR Median IQR Median IQR Median IQR10000 4 2.4-5.2 3.6  3-5.6 9.6  8-11.5 10.5  9.3-13.5 3000 4.1 2.1-5.13.3 2.3-5.7 9.8 8.8-11.5 10.3   9-12.4 1000 4.1  3-5.2 3.6 3.2-5.7 10.1 9-11.7 10.1  9.6-12.6 300 4 3.2-5.1 3.6 3.42-5.5  10.6 9.3-11.8 1110.3-13.1 100 4.7 3.1-5.5 4.3 3.3-5.5 10.8 9.2-16.4 10.6 10.1-14.3 104.1 2.3-6  3.6 2.6-6.5 11.6 11.5-17.1  11.6 10.7-17.5 0 4.8 2.5-8.9 4.6 3.9-10.4 15.6 14.0-22 1  15.6 13.1-24.5

TABLE 6 TGT on Citrated PPP with CTI - SHB PPP (n = 5) ETP[nM/min]Peak[nM] ARC19499 Fresh Frozen Fresh Frozen ng/ml Median IQR Median IQRMedian IQR Median IQR 10000 1484 1129-1592 1736 1312-1850 74.3 70.5-117 106.1 77-122 3000 1360.7 1074-1459 1635 1229-1702 70.5  59-102 97.975-111 1000 1217.5  949-1435 1587.5 1200-1667 68.4 57.1-98.2 94.6 74-107300 1167  893-1353 1429  999-1493 62.1 53.5-97.2 74.3 61-104 100 1075 671-1228 1154.5  850-1340 58.5 40.7-85  60.1 50-92  10 787.5 431-852794.9 550-981 40 21.7-46.6 41 32-64  0 403 186-473 642 189-722 14.9 9.2-25.5 29.3 9-35 Lag time [min] TT peak[min] ARC19499 Fresh FrozenFresh Frozen ng/ml Median IQR Median IQR Median IQR Median IQR 10000 64.7-6.3 5.3 4.9-6.4 12 11.3-14.8 13.7   12-14.5 3000 6 4.7-6  5.3  5-5.713.3 10.9-15.7 12.7 11.8-4.3 1000 5.3 4.6-6.3 5.5 4.5-6.5 12 10.1-14.612.3 11.4-4.4 300 5.5 4.9-5.9 5.7 5.3-5.8 12.7  9.8-14.5 12   11-13.8100 5.3 4.7-6.2 5.5 5.1-6  12.3  9.9-15.1 12.5 10.9-3.7 10 5.7 4.9-6.85.3 5.1-6.5 12.7 11.1-19.7 13.3 11.2-4.3 0 7.8 6.4-9.6 7.2 5.8-8.8 19.814.2-23.4 16.7 14.6-8.8

TABLE 7 TGT on Citrated PPP with CTI - Normal PPP (n = 10) ETP[nM/min]Peak[nM] ARC19499 Fresh Frozen Fresh Frozen ng/ml Median IQR Median IQRMedian IQR Median IQR 10000 1696 1464-2286 2018 1455-2395 257 227-286265 197-274 3000 1903 1788-2424 1897 1746-2356 245 205-270 261 210-2721000 1680 1545-2072 1901 1283-2374 227 188-290 229 182-263 300 19641533-2245 1903 1548-2245 225 199-245 217 183-288 100 1973 1529-2231 17301512-2266 230 200-238 230 189-287 10 2087 1530-2125 2092 1611-2189 239204-274 220 204-269 0 1847 1517-2329 2159 1745-2462 192 161-298 241182-300 Lag time [min] TT peak[min] ARC19499 Fresh Frozen Fresh Frozenng/ml Median IQR Median IQR Median IQR Median IQR 10000 4.3 3.7-4.6 3.4 3-3.5 7.9 6.7-8.9 8.1 7.2-9.4 3000 3.8 3.6-4.5 3.3 3.1-3.9 7.9 7.5-8.87.9 7.5-8.4 1000 4 3.5-4.6 3.5 3.3-3.9 8 7.7-9  7.0 7.4-8.7 300 43.5-4.7 3.5 3.2-3.9 8.3 7.9-9.3 8.2 7.3-9  100 3.9 3.3-4.4 3.5 3.1-3.98.7 7.9-8.9 8.5   6-10.3 10 3.7 3.4-5.1 4 3.2-5  9  7.9-10.8 9.1 7.2-10 0 4.5 3.7-4.6 3.8 3.2-4.7 9.5  7.1-10.4 9.2 7.5-9.9

Example 28

This example demonstrates that ARC19499 can improve clotting in wholeblood and plasma samples from hemophilia A and hemophilia B patients, asmeasured by thromboelastography (TEG).

Blood was collected into 3.2 mL Vacuette tubes containing 3.2% sodiumcitrate and 250 μL 1.3 mg/mL corn trypsin inhibitor (CTI); the final CTIconcentration was 100 μg/mL. Samples were collected from patients withsevere (<1% FVIII; n=10), moderate (1-5% FVIII; n=7) and mild (5-40%FVIII; n=5) hemophilia A, patients with severe hemophilia B (<1% FIX,n=5) and healthy volunteers (n=10). To prepare platelet poor plasma(PPP) for CAT assays, tubes were centrifuged at 2500×g for 15 minutes,the supernatant transferred to fresh Eppendorf tubes, then centrifugedagain at 11000×g for 5 minutes. Plasma was either used immediately orfrozen at −80° C. for later use. To analyze the effects of ARC19499 onthrombin generation, ARC19499 was added to plasma at concentrations of10, 100, 300, 1000, 3000 or 10,000 ng/mL (0.9, 9.0, 27.1, 90.3, 271 or903 nM).

Thromboelastography (TEG) assays were performed using a TEG 5000 seriesinstrument from Haemoscope. Whole blood TEG assays were performed byadding 300 μL whole blood to 40 μL 9 pM tissue factor (TF) and 20 μL 0.2M CaCl₂ in a disposable reaction cup. Amplitude versus time traces wereanalyzed to obtain the R-time (length of time to initiate clotformation, amplitude=2 mm), K-value (measure of the speed of clotformation, equal to the time required to reach an amplitude of 20 mm)and the angle (another measure of the speed of clot formation,calculated from the tangent of the amplitude tracing drawn with itsorigin set to the R-time). Plasma TEG assays were performed similarly,except that supplementary phospholipids (PL) were included. In theseassays 300 μL PPP was mixed with 10 μL 38 pM TF, 30 μL 48 μM PL (20%phosphatidyl serine, 20% phosphatidyl ethanolamine, 60% phosphatidylcholine; Avanti Polar Lipids) and 20 μL 0.2 M CaCl₂. The final PLconcentration in these reactions was 4 μM.

FIGS. 100-103 show representative whole blood TEG data from a healthyvolunteer (HV), a patient with severe hemophilia A (SHA), a patient withmoderate hemophilia A (MoHA) and a patient with mild hemophilia A(MiHA). Median data for healthy volunteers, all three hemophilia Apatient groups, and the severe hemophilia B patient group are shown inFIG. 104. The baseline R-time values were elevated in all hemophiliapatient groups compared to healthy controls, indicating a delay in clotinitiation, with the most significant effect observed in the severehemophilia A and B samples. Additionally, the K-values were increasedand angles decreased in hemophilia patient samples compared to healthycontrols. Both of these effects indicate less rapid clot formationcompared to normal, although the effects were similar in all patientgroups regardless of the severity of factor deficiency.

As shown in the individual (FIGS. 101-103) and median data plots (FIG.104), the addition of ARC19499 improved clot formation in whole blood,as measured by TEG. Increasing concentrations of ARC19499 (up to 10,000ng/mL, 903 nM) substantially normalized all of the TEG parameters in allof the patient groups (FIG. 104). ARC19499 restored normal clotinitiation (R-time) and development (K value and angle).

Similar results were observed in plasma TEG assays. Individual plasmaTEG data from representative patients with severe hemophilia A (SHA),moderate hemophilia A (MoHA) mild hemophilia A (MiHA) are shown in FIGS.105-107, and the median data are presented in FIG. 108. Baselineclotting effects in hemophilia plasma samples showed a trend towardcorrelation with disease severity in all three TEG parameters, with themost substantial effects observed for severe hemophilia A and B samples(FIG. 108). The addition of ARC19499 improved all three parametersdescribing clot formation. Increasing concentrations of ARC19499appeared to normalize clotting in all of the patient groups, as measuredby R-time and K-value. In contrast, while ARC19499 appeared to normalizethe angle in both mild and moderate hemophilia A groups, the angle inthe severe hemophilia A and B groups did not fully correct even at thehighest ARC19499 concentration tested. Nevertheless, substantialimprovement was observed in all of the groups.

Median data with interquartile ranges are presented for whole blood TEGmeasurements in the following tables: Table 8, severe hemophilia A(SHA); Table 9, moderate hemophilia A (MoHA); Table 10, mild hemophiliaA (MiHA); Table 11, severe hemophilia B (SHB); and Table 12, normal.Additional tables show median data with interquartile ranges for plasmaTEG measurements: Table 13, severe hemophilia A (SHA); Table 14,moderate hemophilia A (MoHA); Table 15, mild hemophilia A (MiHA); Table16, severe hemophilia B (SHB); and Table 17, normal. In all of thetables, the expected normal range for each parameter is shown in italicsin the column heading. Taken together, the data show that ARC19499improves clot formation in hemophilia A patient plasma of all severitylevels, and in severe hemophilia B plasma.

TABLE 8 TEG on Citrated Blood with CTI - SHA (n = 10) R time K Angle ARC[min] [min] [deg] 19499 10.6-15.6 1.2-14.8 32.1-56.7 (ng/ml) Median IQRMedian IQR Median IQR 10000 11.1 10.2-18.3 4 3.3-6.6 49.6 33.3-54.2 300015.2 11.2-20.4 5.3 3.6-7.5 38.7 31.2-50.8 1000 16.4 13.5-21.8 6.84.4-9.0 35.7 25.6-47.5 300 19.7 16.4-29.0 8.2  5.4-10.4 30.7 23.6-36.3100 25.3 19.6-33.0 9  6.3-12.6 25.8 17.1-33.1 10 31.9 25.9-50.9 14.711.3-25.3 19.5 11.0-20.1 0 53 43.8-73.0 23 15.8-29.9 8.2  5.0-13.15

TABLE 9 TEG on Citrated Blood with CTI - MoHA (n = 7) R time K Angle ARC[min] [min] [deg] 19499 5.9-16.3 1.7-5.3 48.5-59 (ng/ml) Median IQRMedian IQR Median IQR 10000 9  4.5-12.4 3.8 3.2-4.8  54.1 51-59 300010.7  7.7-16.1 4.7 3.6-6.1  45.3 43.6-52  1000 11.6 6.75-17  6 3.9-10.443 29.7-50.3 300 14.1  6.0-16.2 6.7 3.8-10.8 42.5 30.4-48.7 100 14.310.2-17.8 7 6.0-9.2  35.9 29.1-40.1 10 18 11.2-21.5 7.4 4.0-10.1 27.726.6-37.8 0 28.1 21.7-37.5 17.6 6.8-20.8 17.1  9.3-23.6

TABLE 10 TEG on Citrated Blood with CTI - MiHA (n = 5) R time K AngleARC [min] [min] [deg] 19499 5.9-16.3 1.7-5.3 48.5-59 (ng/ml) Median IQRMedian IQR Median IQR 10000 7.3 5.5-13.8 4.7 2.7-6.8  48.7 38.4-62.83000 7.8 6.5-17.2 8.3 6.4-12.4 42.8 30.0-47.6 1000 8.6 7.9-14.5 11.88.0-12.6 41.5 22.0-48.9 300 8.9 7.8-23.1 12.1 9.7-18.5 39.1 21.4-41.0100 13.3 10.5-24.9  16.6 9.3-22.4 26.8 13.0-41.2 10 16.8 11.8-38.7  1910.0-32.0  16.5 11.8-30.0 0 22 15.9-46.1  19 10.8-23.8  9.7  5.5-28.0

TABLE 11 TEG on Citrated Blood with CTI - SHB (n = 5) R time K Angle ARC[min] [min] [deg] 19499 5.9-16.3 1.7-5.3 48.5-67.9 (ng/ml) Median IQRMedian IQR Median IQR 10000 13.5 10.3-16.9 4.9 4.2-6.4 40.3  36-46.63000 15.2 12.2-18.6 5.5  5-6.4 41.9 36.3-42.6 1000 20.3 17.6-21.2 6.3 5.4-10.2 30.7 21.6-39.6 300 24.6 22.3-31.8 10.3   8-15.5 24.7 17.9-28.9100 30.4 24.9-34.9 12.5 9.5-14  17 14.1-24.9 10 35.8 30.3-39.2 13.711.8-16.3 13.7 11.8-16.3 0 41.6 39.9-57.8 16 14.4-31.8 10.04  6.2-12.5

TABLE 12 TEG on Citrated Blood with CTI - HV (n = 10) R time K Angle ARC[min] [min] [deg] 19499 5.9-16.3 1.7-5.3 48.5-67.9 (ng/ml) Median IQRMedian IQR Median IQR 10000 9.3 8.7-10.3 2.7 2.1-3.5 58.2 51.7-61.6 30008.5 8.3-11.2 3.2 2.3-4.0 58.9 56.7-60.9 1000 8.8 8.4-10.5 2.8 2.2-3.257.8 54.8-63.6 300 9.3 8.4-11.2 2.9 2.6-3.4 57.8 54.2-61.2 100 9.97.2-12.2 2.8 2.3-4.5 58.5 50.0-62.6 10 10.4 9.4-11.5 2.8 2.3-3.1 57.152.0-62.6 0 12 8.6-12.9 3.2 2.8-4.2 56 51.6-57.9

TABLE 13 TEG on Citrated PPP with CTI - SHA (n = 10) R time K Angle ARC[min] [min] [deg] 19499 10.6-15.6 1.2-14.8 32.1-56.7 (ng/ml) Median IQRMedian IQR Median IQR 10000 10.3  8.9-21.5 10  7.5-14.7 32.3 24.1-38.53000 14.2 10.4-23.8 15.4 10.9-17.3 22.3 16.5-25.7 1000 15 15.7-25.7 15.112.1-19.0 21.2 18.8-23.9 300 15.7  9.8-29.5 19  7.8-21.0 14.9 11.6-16.8100 15.8 14.3-34.1 19.6 18.3-21.0 14 10.7-14.4 10 23.6 15.7-29.8 2019.8-23.0 9.9  8.8-10.7 0 32.6 25.5-55.4 24.1 20.4-27.3 6.1 4.4-7.9

TABLE 14 TEG on Citrated PPP with CTI - MoHA (n = 7) R time K Angle ARC[min] [min] [deg] 19499 10.6-15.6 1.2-14.8 32.1-56.7 (ng/ml) Median IQRMedian IQR Median IQR 10000 13.8 11.5-15.2 7.3 4.9-9.9  40.4 35.8-45.63000 16.8 10.6-21  8.5 5.9-8.9  35.6 30.9-40.2 1000 17.9  12-22.1 8.75.1-10.1 32 27.1-36.2 300 18.5 12.6-22.5 10.4 7.4-12.3 23 17.3-29.1 10019.1 11.9-23.5 11.5 9.3-14.4 20.7 19.2-24.6 10 20.9 13.8-25.6 11.99.6-14.4 15.2 13.1-18.7 0 23.6 18.2-28.1 16.5 13-18  12 7.9-14 

TABLE 15 TEG on Citrated PPP with CTI - MiHA (n = 5) R time K Angle ARC[min] [min] [deg] 19499 10.6-15.6 1.2-14.8 32.1-56.7 (ng/ml) Median IQRMedian IQR Median IQR 10000 11.8 3.4-11.9 3.3 1.3-3.8 56.6 53.3-73.93000 11.8 5.2-13.9 3.4 2.0-4.0 52.6 51.3-66.4 1000 12.8 4.0-14.9 3.42.3-4.9 50.6 47.8-62.0 300 14 3.7-18.6 3.7 3.5-3.8 46.4 44.1-52.6 10014.7 5.6-21.7 5 3.8-6.2 37.9 34.5-41.1 10 15.8 13.9-22.0  7.4  4.2-10.129.1 23.5-35.1 0 19.8 17.7-26.9  9.6  4.6-12.2 21.1 16.2-35.0

TABLE 16 TEG on Citrated PPP with CTI - SHB (n = 5) R time K Angle ARC[min] [min] [deg] 19499 10.6-15.6 1.2-14.8 32.1-56.7 (ng/ml) Median IQRMedian IQR Median IQR 10000 10.2  8.2-14.3 7.7  4.8-17.7 31.2 20.6-49.93000 12.3  9.7-16.9 14.5  6.5-23.2 26.6 17.2-24.1 1000 19.2 12.7-21.8 23 8.5-26.5 22.4 12.1-30.4 300 21 12.5-28.5 24.2 14.3-28.8 12.5  5.2-20.4100 22.6 13.6-31.6 19.6 16.8-27.4 5.3  4.9-15.3 10 33.1 16.9-38.3 19.117.1-28.4 4.8  3.7-14.1 0 36.7 27.9-44.5 22.5 20.5-59.9 2.6 1.5-7.6

TABLE 17 TEG on Citrated PPP with CTI - HV (n = 10) R time K Angle ARC[min] [min] [deg] 19499 10.6-15.6 1.2-14.8 32.1-56.7 (ng/ml) Median IQRMedian IQR Median IQR 10000 9.8  6.7-13.2 4.8 3.5-6.4  51.2 50.0-53.23000 8.7  7.0-10.7 7.8 5.2-10.8 45 44.0-49.9 1000 11.6 11.0-12.1 10.35.8-10.5 40.9 39.9-42.0 300 12.7 12.1-13.8 10.4 6.5-11.3 37.7 36.5-40.3100 14.3 13.1-15.3 10.1 5.9-11.2 39.3 39.6-49.0 10 15 13.1-15.7 11.710.1-11.9  38.8 38.2-44.7 0 12.7 12.1-14.3 7.9 4.9-11.3 40.3 38.5-48.3

Example 29

This example demonstrates that the in vitro activity of ARC19499 can bereversed.

Four reversal agents (ARC23085, ARC23087, ARC23088 and ARC23089) weremixed with ARC19499 and tested in both the calibrated automatedthrombogram (CAT) and the thromboelastography (TEG®) assays inhemophilia A plasma (FIG. 109).

For the CAT assay, ARC19499 was incubated with each reversal agentindividually for 5 minutes at 37° C. The mixture was then added tohemophilia A plasma at a final concentration of 100 nM ARC19499 andincreasing concentrations of ARC23085, ARC23087, ARC23088, or ARC23089(2.5, 5, 10, 20, 40, 80, 160, and 320 nM). The CAT assay was thenperformed as previously described using a final TF concentration of 1.0pM. ARC19499 alone improved the ETP of hemophilia A plasma from ˜600 nMto ˜900 nM (FIG. 109A). All four tested reversal agents blocked thisimprovement when tested at concentrations>80 nM. ARC23085 and ARC23089almost completely reversed ARC19499 activity at 160 nM, while ARC23087and ARC23088 almost completely reversed ARC19499 activity at 320 nM.Looking at peak thrombin (FIG. 109B), the four reversal agents showed asimilar partial reversal of ARC19499 activity at 80 nM. Again, by 160nM, ARC23085 and ARC23089 completely reversed ARC19499 activity, whilethe other two reversal agents did so by 320 nM.

For the TEG® assay, ARC19499 and one of each of the four reversal agentswere mixed with hemophilia A plasma, and clotting was initiated with theaddition of TF and CaCl₂. This was performed with and without a 5 minutepreincubation of ARC19499 and the additional reversal agent at 37° C.When ARC19499 was tested alone, the aptamer corrected the prolongedR-value of hemophilia A plasma from 54 minutes to 8.3 minutes (FIG.109C). ARC23085 partially reversed this improvement with R-values of 30and 24 minutes with and without the 5 minute preincubation,respectively. ARC23087 demonstrated no ability to reverse ARC19499 whenthere was no preincubation, but with the incubation it did reverseARC19499 activity, resulting in an R-value of 38 minutes. ARC23088demonstrated little to no ability to reverse ARC19499 activity in thisassay, independent of any preincubation. Similar to ARC23087, ARC23089showed little ability to reverse ARC19499 activity when there was nopreincubation. But, when the reversal agent was incubated with ARC19499prior to the assay, it reversed ARC19499 activity almost completely,resulting in an R-value of 42 minutes (FIG. 109C).

These experiments indicate that the in vitro activity of ARC19499 can bereversed.

Example 30

This example demonstrates that ARC19499 does not inhibit the in vitroanti-coagulant activity of low molecular weight heparin (LMWH).

In this assay, increasing concentrations of ARC19499 and increasingconcentrations of LMWH were mixed together and added to hemophilia Aplasma. The thrombin generation ability of these plasma mixtures wasanalyzed using the calibrated automated thrombogram (CAT) assay. FIG.110 shows the thrombin generation curves of the increasing aptamerconcentrations in the presence of each LMWH concentration. ARC19499 wastested at 0.1, 1, 10, 100 and 1000 nM. LMWH was tested at 0 (FIG. 110A),0.156 (FIG. 110B), 0.312 (FIG. 110C), 0.625 (FIG. 110D), 1.25 (FIG.110E), 2.5 (FIG. 110F) and 5.0 IU (international units)/mL (FIG. 110G).

In FIG. 111A, the ETP values for each combination were plotted along they-axis, with the LMWH concentrations on the x-axis. The same was truefor peak thrombin values in FIG. 111B. In both cases, therapeutic dosesof LMWH (0.5-1.0 IU/mL) strongly inhibited thrombin generation, andhigher concentrations almost completely prevented any thrombin frombeing generated, even in the presence of up to 1000 nM ARC19499 (FIG.111). IC₅₀ values of 381±24.3 nM and 299±18.0 nM for LMWH werecalculated from the ETP and peak thrombin data, respectively, in theabsence of ARC19499, and these results are consistent with previousmeasurements (Robert et al., Is thrombin generation the new rapid,reliable and relevant pharmacological tool for the development ofanticoagulant drugs?, Pharmacol Res. 2009; 59:160-166). Increasingconcentrations of ARC19499 did not appear to significantly alter theIC₅₀ of LMWH (FIG. 112), indicating that ARC19499 does not interferewith the anticoagulant activity of LMWH.

This experiment indicates that even in the presence of 1000 nM ARC19499,therapeutic doses of LMWH still remain anti-coagulant in an in vitroassay.

Example 31

This example demonstrates that the TFPI aptamers are stable to serumnucleases.

In this experiment, 50 μM of each aptamer was incubated in 90% pooledhuman, cynomolgus monkey or rat serum for 72 hours at 37° C. Sampleswere analyzed by HPLC and the percent remaining as a function ofincubation time was determined, as shown in FIG. 113.

Both ARC19498 and ARC19499 were >95% stable over the course of 72 hoursin human, monkey and rat serum (FIGS. 113A and 113B).

ARC19500 was >92% stable over the course of 72 hours in human, monkeyand rat serum (FIG. 113C), and ARC19501 was >80% stable over the courseof 72 hours in human, monkey and rat serum (FIG. 113D).

ARC19881 was >78% stable over the course of 72 hours in human, monkeyand rat serum (FIG. 113E), and ARC19882 was >91% stable over the courseof 72 hours in human, monkey and rat serum (FIG. 113F).

Example 32

This example demonstrates that the TFPI aptamers have biologicalactivity.

In this experiment, a non-human primate model of hemophilia A wascreated by injecting cynomolgus monkeys with a single intravenous (IV)bolus of sheep polyclonal antibody against human Factor VIII (20 mg;50,000 Bethesda Units). 3.5 hours after the IV injection, the monkeyswere treated with either saline (1 mL/kg), recombinant Factor VIIa(rFVIIa) (NovoSeven®; 90 μg/kg bolus) or ARC19499 (either a 600 μg/kg,300 μg/kg or 100 μg/kg bolus). Citrated blood samples were acquiredbefore antibody administration (baseline), 2.5 hours after antibodyadministration, 15 minutes after drug/saline treatment (time=3.75hours), and 1 and 2 hours after drug/saline treatment (time=4.5 and 5.5hours, respectively). Blood was processed to generate plasma, andcitrated plasma samples were assayed for prothrombin time (PT),activated partial thromboplastin time (aPTT), Factor VIII function andthromboelastography (TEG®). The saline-treated monkey received atreatment of ARC19499 (600 μg/kg) after the 5.5 hour time point wasdrawn. Fifteen minutes after receiving this treatment, another citratedblood sample was drawn and processed for plasma.

In order to ensure that ARC19499 inhibits monkey TFPI, plasma taken frommonkeys after antibody treatment were mixed, ex vivo, with increasingconcentrations of ARC19499 from 1 to 1000 nM, and tested in a TEG® assay(FIG. 114). The plasma mixed with aptamer was compared to plasma withoutaptamer, both before and after antibody treatment (solid and dashedlines, respectively). Antibody treatment alone prolonged the R-value.The addition of ARC19499 to this antibody-treated plasma corrected theR-value to near baseline levels, suggesting the aptamer wascross-reactive with monkey plasma.

In the plasma sample withdrawn 2.5 hours after antibody injection,Factor VIII levels were below 0.6% and remained there for the course ofthe 5.5 hour assay (FIG. 115). As expected, the PT, which is insensitiveto FVIII, remained unchanged after antibody administration (FIG. 116A);however, upon injection of rFVIIa, there was a slight dip in PT valuesfrom 13.0±0.4 to 10.7±0.4 seconds. aPTT values increased afteradministration of the Factor VIII antibody (FIG. 116B). As seen in thePT assay, rFVIIa administration resulted in a decrease in clotting timein the aPTT assay. There was no change to the aPTT values upon salinetreatment. ARC19499 treatment (at all concentrations) resulted in aprolongation in aPTT. The saline-treated animal that received anARC19499 bolus after the 5.5 hour time point also showed a prolongationin aPTT after aptamer administration. The effect of ARC19499 treatmenton clot development in this hemophilia A-like model was assayed usingtissue factor (TF)-activated TEG®. In all animals tested, antibodyadministration resulted in a prolongation in R-value (FIG. 117A). Salinetreatment had no further effect on the R-value, while both rFVIIa andthe 600 μg/kg and 300 μg/kg ARC19499 treatments resulted in a decreasein R-value to levels close to baseline. The 100 μg/kg dose of ARC19499was an ineffective dose and did not have a positive effect on theR-value. In the saline-treated monkey, the additional injection ofARC19499 at the end of the study had an immediate effect on decreasingthe R-value. The angle (FIG. 117B) and maximum amplitude (MA) (FIG.117C) were both reduced after antibody administration. The 600 μg/kg and300 μg/kg ARC19499 treatments appeared to have similar increasingeffects on the angle as NovoSeven®; whereas, the 100 μg/kg ARC19499treatment behaved similarly to the saline treatment (FIG. 117B). All ofthe treatments, including saline, appeared to have no additional effecton MA (FIG. 117C).

In a second set of related experiments, monkeys were treated with thesame concentration of anti-FVIII antibody followed by 300 μg/kg ARC19499or 90 μg/kg NovoSeven® 1 hour later. Citrated blood samples wereacquired and processed for plasma at baseline, 60 minutes after antibodyadministration, and 60, 120, 180, 240, 300 and 420 minutes after drugadministration. Plasma samples were tested using the TF-activated TEG®assay as described above. As before, antibody administration resulted inincreased R-values (FIG. 118A), and decreased angle and MA values (FIG.118B-C). ARC19499 and NovoSeven® behaved in very similar manners,restoring the R-values to near-baseline levels and improving both theangle and MA values (FIG. 118).

Taken together, the Factor VIII, aPTT and TEG® all indicate thesuccessful induction of a hemophilia A-like state in these monkeys. Themoderate prolongation of the aPTT upon injection of ARC19499 most likelyreflects further, non-specific inhibition of the intrinsic cascade,which has been previously observed with other aptamers. However, theclear correction of the clot time (R-value) in TF-activated plasmameasured by the TEG® assay suggests that the hemostatic defect due toloss of Factor VIII was successfully bypassed by ARC19499 inhibition ofTFPI.

An additional observation of ARC19499 treatment was that it appeared toresult in an increase in plasma levels of TFPI. In a preliminaryexperiment, TFPI concentrations in samples from these cynomolgusexperiments mentioned above, following ARC19499 treatment, exceeded theupper limit of quantitation of the TFPI ELISA when diluted 1:40. Thiseffect was analyzed more closely by measuring the TFPI levels inEDTA-plasma samples from cynomolgus monkeys that received a very highdose (20 mg/kg) of ARC19499 either via IV or subcutaneous (SC)administration. Blood samples were drawn periodically over two weeks,which enabled the assessment of changes in TFPI plasma concentrationsfollowing a more prolonged exposure to ARC19499. TFPI concentrationswere measured in serially diluted plasma samples (diluted up to 1:1600)at the following timepoints: pre-dose, 0.083 (IV only), 0.5, 2, 6, 12,36, 72, 120, 168, 240 and 336 hours post-dose. In animals treated witheither an IV or SC dose of ARC19499, TFPI levels increased immediatelyfrom ˜0.2 nM (pre-dose) to 5-7 nM at the first time point, then moregradually over time to peak levels of 91±12 and 71±2 nM, respectively(FIG. 119). The pattern of TFPI levels was similar in the monkeystreated with either the IV or SC dose, increasing to peak levels at72-120 hours followed by a gradual decline. However, the ARC19499t_(max) estimates were 5 minutes and 24 hours for the IV and SC doses,respectively (data not shown). Thus, although ARC19499 stimulated anincrease in plasma TFPI in vivo, the changes in TFPI and ARC19499concentrations over time do not necessarily correlate. The mechanism forthe increase in TFPI is unknown, but it is possible that the initialexposure to ARC19499 caused the rapid release of TFPI from theendothelial surface while subsequent increases were due to slow releaseof TFPI from intracellular stores, upregulation of TFPI expression,and/or inhibition of TFPI clearance mechanisms. This increase in plasmaTFPI levels upon aptamer treatment was specific for ARC19499. An aptameragainst a target other than TFPI did not appear to increase TFPI levels,especially not to the same level as ARC19499 (data not shown).

Example 33

This example demonstrates that ARC19499 shortens bleeding time in anon-human primate (NHP) model of hemophilia A and that this shortenedbleeding time is reflected in most cases by a concomitant correction inthe thromboelastography (TEG®) R-value (a measure of the time to initialclot formation) from the prolonged R-value associated with hemophilia Aand present in the NHP model described below.

Bleeding time, with and without ARC19499 treatment, was assessed in aNHP model of hemophilia A in which cynomolgus monkeys were dosed with ananti-FVIII antibody (FVIII Ab) to induce a hemophilia A-like state. Asdiagramed in FIG. 120, bleeding time was measured at the beginning ofthe experiment (baseline), and then 2.5 hours after FVIII Abadministration. Treatment with 1 mg/kg bolus doses of ARC19499 were thenbegun. Bleeding time was measured 1 hour after the first ARC19499 dosefor all groups of monkeys. For those monkeys whose bleeding times hadnot corrected, bleeding time was again assessed 17 minutes after thesecond ARC19499 dose. For those monkeys whose bleeding times still hadnot corrected, bleeding time was again assessed 17 minutes after thethird ARC19499 dose (groups 3 and 4).

For bleeding time assessment and collection of blood samples, a monkeywas anesthetized, and a line was placed in the femoral vein for bloodpressure determinations and the saphenous vein on the opposite side fromthe saphenous vein used for the bleeding time assessment wascatheterized with a 22 gauge catheter for dosing and sampling. Theschedule for bleeding time assessment and related FVIII Ab and ARC19499dosing and blood sampling are indicated in FIG. 120. A pre-treatmentbaseline blood sample and a bleeding time (pre-treatment bleeding time,BT₀) were taken 10 and 5 minutes, respectively, before dosing with FVIIIAb. Blood samples were taken by first withdrawing approximately 3-4 mLof blood mixed with saline, which was then set aside. An undiluted bloodsample was then taken using a syringe. The collected blood wasimmediately injected into citrate-containing tubes and inverted tentimes to mix. A sample of the citrated whole blood was analyzed by TEG®and the remaining citrated blood was processed to obtain plasma foranalysis. The first blood-saline sample was then injected back into themonkey, followed by a flush of saline equivalent to the volume of bloodsample taken.

The saphenous vein was exposed for the bleeding time assessment. A 22gauge 1 inch hypodermic needle (Kendall Monoject, Kendall Healthcare,Mansfield, Mass.) was carefully bent at the bevel to a 90° C. angleusing a hemostat; the bend was 5 mm from the tip of the needle. Theneedle was inserted into the exposed vein up to the bend in the needle,puncturing only one wall of the vein. Blood was wicked away from thevein using Surgicutt Bleeding Time Blotting Paper (ITC, Edison, N.J.);care was taken to not touch the actual puncture site. Time was measuredfrom the moment bleeding began until cessation of bleeding, asdetermined by inability to wick away blood. Blood pressure readings weretaken one to five minutes before and after bleeding time assessment.After bleeding time assessment, the wound was closed with Gluture(Abbott Labs, Abbott Park, Ill.).

FVIII Ab (sheep anti-human FVIII polyclonal antibody, Lot Y1217, fromHaematologic Technologies Inc (Essex Junction, Vt.)) was administered tomonkeys intravenously (IV) as a single slow bolus dose via a 22 gaugecatheter placed in the saphenous vein on the opposite side from thesaphenous vein used for the bleeding time assessment. It was kept frozenat −80° C. until use, and was thawed and re-frozen no more than 3 timesbefore use. Each monkey received 12,642 Bethesda units/kg of FVIII Ab.Two hours after the FVIII Ab was given, another blood sample was taken;30 minutes later the bleeding time was assessed (post FVIII bleedingtime, BT_(FVIII)). A first dose of ARC19499 was administered IV as asingle slow bolus. In all cases prior to dosing of either the FactorVIII Ab or ARC19499, 0.5-1.0 mL of fluid were removed from the catheter,the FVIII Ab or ARC19499 was administered, and the catheter was thenflushed with 2-3 mL warm saline. A blood sample was taken 55 minuteslater. One hour after the administration of the first ARC19499 dose, thebleeding time was assessed (post first ARC19499 dose bleeding time,BT₁). Determination was then made as to whether the bleeding time hadbeen corrected by the first dose of ARC19499. Correction was consideredto have been attained if the difference between the pre-treatmentbaseline and post first ARC19499 dose bleeding times was less than halfthe difference between the pre-treatment baseline and post FVIIIbleeding times (i.e., (BT₁−BT₀)/(BT_(FVIII)−BT₀)<0.5). If the bleedingtime was successfully corrected, the bleeding time study was completedfor that animal. If the bleeding time was not corrected, a second doseof 1 mg/kg ARC19499 was administered ten minutes after the initiation ofthe prior bleeding time assessment. A blood sample was taken 12 minuteslater; a bleeding time assessment was begun 17 minutes after the secondARC19499 injection (post second ARC19499 dose bleeding time, BT₂).Determination was then made as to whether the bleeding time had beencorrected by the second dose of ARC19499. If the bleeding time wassuccessfully corrected, the bleeding time study was completed for thatanimal. If the bleeding time was not corrected, a third dose of 1 mg/kgARC19499 was administered ten minutes after the initiation of the priorbleeding time assessment. A blood sample was taken 12 minutes later; ableeding time assessment was begun 17 minutes after the second ARC19499injection (post third ARC19499 dose bleeding time, BT₃). Determinationwas then made as to whether the bleeding time had been corrected by thethird dose of ARC19499. Regardless of whether the bleeding time wassuccessfully corrected, the bleeding time study was completed for thatanimal. If the bleeding time was not corrected, a fourth dose of 3 mg/kgARC19499 was administered 14 minutes after the initiation of the priorbleeding time assessment; a blood sample was taken for whole blood TEG®analysis but no further bleeding times were assessed due to lack ofavailable vein of type similar to that used in the study. AdditionalARC19499 (1 to 3 mg/kg) was administered prior to recovery of eachanimal as a precaution against bleeding over the next 24 hours. Theactual time points for blood sampling, compound dosing and bleeding timeassessment for each animal are within 5 minutes of the time pointreported.

FVIIIa activity levels in citrated plasma from monkeys in this studywere measured using the Coamatic FVIII assay from Chromogenix(Diapharma, Columbus Ohio). Samples acquired during the study werecompared to a standard curve generated from a pool of the pre-treatmentbaseline plasma samples. All samples and standards were diluted 80× inreaction buffer provided by the kit. The reaction was carried out as perthe manufacturer's instructions, reading the change in absorbance at 405nm over 45 minutes. The FVIII activity levels associated with thecynomolgus monkey plasma samples are shown in Table 18 and FIG. 121(Table 18A and FIG. 121A: Group 1: monkeys whose bleeding times werecorrected with one dose of 1 mg/kg ARC19499; Table 18B and FIG. 121B:Group 2: monkeys whose bleeding times were corrected with two doses of 1mg/kg ARC19499; Table 18C and FIG. 121C: Group 3: monkey whose bleedingtime was corrected with three doses of 1 mg/kg ARC19499; Table 18D andFIG. 121D: Group 4: monkey whose bleeding time was not corrected withthree doses of 1 mg/kg ARC19499). Prior to antibody treatment, theplasma from all monkeys had very high FVIII activity levels, varyingfrom 57.6% to 93.5% (Table 18). After antibody treatment, this activitydropped to below measurable levels (less than 0.1%) in all animals, andremained low for the course of the assay (FIG. 121). Most importantly,there was no difference noted by this assay in the level of FVIIIinactivation between the different groups of animals, indicating thatthe dose of ARC19499 required to correct the bleeding time was not arelated to differing levels of FVIII activity post FVIII Abadministration.

TABLE 18 FVIII activity levels A. Group 1: Monkeys Whose Bleeding Timeswere Corrected by One Dose of 1 mg/kg ARC19499 Time NHP NHP NHP NHP NHPNHP point 0703333 0704039 0701565 0604313 0703551 0603477 (min) % % % %% % Baseline −10 64.4 57.6 74.1 93.5 71.8 82.4 2 hr post FVIII ab 120−1.6 −2.2 −1.8 −1.5 −1.8 −2.6 treatment 55 min post first 210 −1.5 −2.1−1.8 −2.2 −1.6 −2.3 ARC19499 treatment B. Group 2: Monkeys WhoseBleeding Times were Corrected by Two Doses of 1 mg/kg ARC19499 Time NHPNHP NHP NHP point 0610301 0702277 0611655 0511011 (min) % % % % Baseline−10 63.0 79.6 59.5 89.7 2 hr post FVIII ab 120 −2.1 −0.3 −2.3 −2.0treatment 55 min post first 210 −2.2 −0.1 −2.2 −1.9 ARC19499 treatment12 min post second 237 ns 0.1 −2.2 −1.7 ARC19499 treatment C. Group 3:Monkey Whose Bleeding Time was Corrected by Three Doses of 1 mg/kgARC19499 Time point NHP0702073 (min) % Baseline −10 76.8 2 hr post FVIIIab 120 0.0 treatment 55 min post first 210 −0.2 ARC19499 treatment 12min post second 239 −0.3 ARC19499 treatment 12 min post third 278 0.3ARC19499 treatment D. Group 4: Monkey Whose Bleeding Time was NotCorrected by Three Doses of 1 mg/kg ARC19499 Time point NHP0607367 (min)% Baseline −10 62.5 2 hr post FVIII ab 120 0.8 treatment 55 min postfirst 210 −1.4 ARC19499 treatment 12 min post second 237 −2.0 ARC19499treatment 12 min post third 264 −2.0 ARC19499 treatment 12 min postfourth 296 −1.7 ARC19499 treatment

The mean group bleeding times (±SEM) for Group 1 monkeys (whose bleedingtimes were corrected with one dose of 1 mg/kg ARC19499) are shown inTable 19. The mean group bleeding times for this group are also plottedagainst the time points of the blood samples in FIG. 122 (FIG. 122A: inseconds, FIG. 122B: in terms of % of baseline bleeding time). Treatmentwith the anti-FVIII antibody resulted in a prolongation of the groupmean bleeding time to 203±15% of the baseline group mean bleeding time.Treatment of the monkeys with 1 mg/kg ARC19499 corrected the group meanbleeding time back to essentially baseline levels (102±11% of thebaseline group mean bleeding time). Individual bleeding times for Group1 monkeys are shown in Table 20; the individual monkey bleeding times ateach time point are also plotted in FIG. 123 (FIG. 123A: in seconds,FIG. 123B: in terms of % of baseline bleeding time). All monkeys in thisgroup showed a prolongation of their bleeding times in response toadministration of FVIII Ab compared to their baseline bleeding times(range: 162 to 252% of the baseline bleeding time). All monkeys in thisgroup also exhibited a correction of their bleeding times in response toadministration of 1 mg/kg ARC19499 compared to their baseline bleedingtimes (range: 82 to 152% of the baseline bleeding time).

TABLE 19 Mean Bleeding Times in Monkeys Whose Bleeding Times wereCorrected by One Dose of 1 mg/kg ARC19499 (Group 1) Group 1: MeanBleeding Times Time point Mean SEM (min) (sec) (sec) Baseline −5 41 5.12.5 hr post FVIII ab 150 84 13 treatment 1 hr post 215 40 3.5 ARC19499treatment

TABLE 20 Individual Bleeding Times in Monkeys Whose Bleeding Times wereCorrected by One Dose of 1 mg/kg ARC19499 (Group 1) Group 1: IndividualBleeding Times Time NHP NHP NHP NHP NHP NHP point 0703333 07040390701565 0604313 0703551 0603477 (min) (sec) (sec) (sec) (sec) (sec)(sec) Baseline −5 36 23 45 42 41 61 2.5 hr post FVIII ab 150 62 58 85 6885 145 treatment 1 hr post 215 31 35 37 47 38 54 ARC19499 treatment

The mean group bleeding times (±SEM) for Group 2 monkeys (whose bleedingtimes were corrected with two doses of 1 mg/kg ARC19499) are shown inTable 21. The mean group bleeding times for this group are also plottedagainst the time points of the blood samples in FIG. 124 (FIG. 124A: inseconds, FIG. 124B: in terms of % of baseline bleeding time). Treatmentwith the anti-FVIII antibody resulted in a prolongation of the groupmean bleeding time to 195±26% of the baseline group mean bleeding time.After treatment of the monkeys with 1 mg/kg ARC19499, the group meanbleeding time did decrease, but only to 175±20% of the baseline groupmean bleeding time. An additional dose of 1 mg/kg ARC19499 subsequentlyreduced the group mean bleeding time to essentially baseline levels(94±17% of the baseline group mean bleeding time). Individual bleedingtimes for Group 2 monkeys are shown in Table 22; the individual monkeybleeding times at each time point are also plotted in FIG. 125 (FIG.125A: in seconds, FIG. 125B: in terms of % of baseline bleeding time).All monkeys in this group showed a prolongation of their bleeding timesin response to administration of FVIII ab compared to their baselinepre-treatment bleeding times (range: 143 to 263% of the baselinebleeding time). After administration of 1 mg/kg ARC19499, three of themonkeys exhibited a slight decrease in their bleeding times while onemonkey showed a small increase in bleeding time. All monkeys in thisgroup exhibited a correction of their bleeding times in response to asecond dose of 1 mg/kg ARC19499 (range: 61 to 138% of baseline bleedingtime).

TABLE 21 Mean Bleeding Times in Monkeys Whose Bleeding Times WereCorrected by Two Doses of 1 mg/kg ARC19499 (Group 2) Group 2: MeanBleeding Times Time point Mean SEM (min) (sec) (sec) Baseline −5 35 122.5 hr post FVIII ab 150 62 13 treatment 1 hr post first 215 59 16ARC19499 treatment 17 min post second 242 31 8.4 ARC19499 treatment

TABLE 22 Individual Bleeding Times in Monkeys Whose Bleeding Times WereCorrected by Two Doses of 1 mg/kg ARC19499 (Group 2) Group 2: IndividualBleeding Times Time NHP NHP NHP NHP point 0610301 0702277 06116550511011 (min) (sec) (sec) (sec) (sec) Baseline −5 29 23 70 19 2.5 hrpost FVIII ab 150 60 38 100 50 treatment 1 hr post first 215 50 33 10744 ARC19499 treatment 17 min post second 242 40 14 50 20 ARC19499treatment

The bleeding times for the Group 3 monkey are shown in Table 23; thebleeding times are also plotted against the time points of the bloodsamples in FIG. 126 (FIG. 126A: in seconds, FIG. 126B: in terms of % ofbaseline bleeding time). This monkey showed a prolongation of itsbleeding time in response to administration of FVIII Ab to 220% of thebaseline bleeding time. In response to treatment with 1 mg/kg dose ofARC19499, the bleeding time increased to 330% of the baseline. Treatmentof this monkey with two doses of 1 mg/kg ARC19499 produced a reductionof the bleed time to 210% of the baseline bleeding time. To confirm thatthis second dose had indeed not corrected the bleeding time, anadditional bleeding time assessment was performed 38 minutes afteradministration of the second ARC19499 dose. This bleeding time of 45seconds was very close to the 42 seconds measured 12 minutes after thesecond dose of ARC19499 was given, confirming that the bleeding time hadnot been corrected by the two doses of ARC19499. An additional dose of 1mg/kg ARC19499 corrected the bleeding time to 85% of the baselinebleeding time.

TABLE 23 Bleeding Times in Monkey Whose Bleeding Time was Corrected byThree Doses of 1 mg/kg ARC19499 (Group 3) Group 3: Bleeding Times Timepoint NHP0702073 (min) (sec) Baseline −5 20 2.5 hr post FVIII ab 150 44treatment 1 hr post first 215 66 ARC19499 treatment 17 min post second243 42 ARC19499 treatment 37 min post second 263 45 ARC19499 treatment17 min post third 282 17 ARC19499 treatment

The bleeding times for the Group 4 monkey are shown in Table 24; thebleeding times are also plotted against the time points of the bloodsamples in FIG. 127 (FIG. 127A: in seconds, FIG. 127B: in terms of % ofbaseline bleeding time). This monkey showed a prolongation of itsbleeding time in response to administration of FVIII Ab to 143% of thebaseline bleeding time. In response to treatment with 1 mg/kg dose ofARC19499, the bleeding time increased markedly to 193% of the baselinebleeding time. The bleeding time after treatment of this monkey with twodoses of 1 mg/kg ARC19499 then decreased to 154% of the baselinebleeding time. An additional dose of 1 mg/kg ARC19499 failed tosignificantly change the bleeding time, which was now 159% of thebaseline bleeding time. No additional bleeding time assessments could bedone on this animal due to a lack sufficient available vein consistentwith that used for previous assessments.

TABLE 24 Bleeding Times in Monkey Whose Bleeding Time was Not Correctedby Three Doses of 1 mg/kg ARC19499 (Group 4) Group 4: Bleeding TimesTime point NHP0607367 (min) (sec) Baseline −5 56 2.5 hr post FVIII ab150 80 treatment 1 hr post first 215 108 ARC19499 treatment 17 min postsecond 243 86 ARC19499 treatment 17 min post third 273 89 ARC19499treatment

The coagulation status of the cynomolgus whole blood was analyzed usingthe TEG® assay on citrated whole blood samples. To initiate the clottingreaction, 330 μL citrated whole blood was added to a disposable cup(Haemonetics Corp, cat no. 6211) containing 20 μL 0.2 M [HaemoneticsCorporation (Braintree, Mass.)] and 10 μL tissue factor (TF) (finaldilution of 1:200000 at 37° C.) Innovin (Dade-Behring, Newark, Del.) wasused as a tissue factor (TF) source, reconstituted in water as permanufacturer recommendation, and diluted 1:5555 in 0.9% saline prior touse. Reconstituted stock Innovin was stored at 4° C. for less than 4weeks. The time to initial clot formation (R-value) was measured usingthe Haemoscope TEG® 5000 system (Haemonetics Corporation, Braintree,Mass.).

The mean group R-values (±SEM) for Group 1 monkeys (whose bleeding timeswere corrected with one dose of 1 mg/kg ARC19499) are shown in Table 25.The mean group R-values are also plotted against the time points of theblood sample (FIG. 128). Treatment with FVIII Ab resulted in aprolongation of the group mean R-value to about 4.8 times the group meanR-value at baseline. While treatment of the monkeys with 1 mg/kgARC19499 reduced the group mean R-value from that obtained after FVIIIAb treatment, this R-value was still about 3.2 times the group meanbaseline R-value. Individual R-values for Group 1 monkeys are shown inTable 26; the individual R-values are also plotted against the timepoints of the blood sample in FIG. 129. All monkeys in this group showeda prolongation of their R-values in response to administration of FVIIIab compared to their baseline pre-treatment R-values (range 2.9 to9.4-fold). All but one of the monkeys in this group also exhibited areduction of their R-values in response to administration of 1 mg/kgARC19499 compared to their baseline pre-treatment R-values (range 1.4 to5.3-fold). One monkey, NHP0701565, showed a slight increase in theR-value after administration of 1 mg/kg ARC19499.

TABLE 25 Mean Whole Blood TEG ® R-values in Monkeys Whose Bleeding Timeswere Corrected by One Dose of 1 mg/kg ARC19499 (Group 1) Group 1: MeanWhole Blood TEG ® R-values Time point Mean SEM (min) (min) (min)Baseline −10 5.6 0.93 2 hr post FVIII ab 120 27 3.3 treatment 55 minpost 210 18 4.8 ARC19499 treatment

TABLE 26 Individual Whole Blood TEG ® R-values in Monkeys Whose BleedingTimes were Corrected by One Dose of 1 mg/kg ARC19499 (Group 1) Group 1:Individual Whole Blood TEG ® R-values Time NHP NHP NHP NHP NHP NHP point0703333 0704039 0701565 0604313 0703551 0603477 (min) (min) (min) (min)(min) (min) (min) Baseline −10 8.5 ns 6.0 2.2 5.0 6.2 2 hr post FVIII ab120 38.6 28.8 34 20.7 22.6 17.8 treatment 55 min post 210 29.4 10.9 35.511.6 9.8 8.4 ARC19499 treatment

The mean group R-values (±SEM) for Group 2 monkeys (whose bleeding timeswere corrected with two doses of 1 mg/kg ARC19499) are shown in Table27. The mean group R-values are also plotted against the time points ofthe blood samples in FIG. 130. Treatment with the anti-FVIII antibodyresulted in a prolongation of the group mean R-value to about 5.9 timesthe group mean R-value at baseline. In response to treatment with 1mg/kg dose of ARC19499, which did not correct the bleed time, the groupmean R-value was reduced to about 4.0 times the baseline group meanR-value. Treatment of the monkeys with two doses of 1 mg/kg ARC19499,which did correct the bleed time in this group, further reduced thegroup mean R-value from that obtained after FVIII Ab treatment, althoughthis R-value was still about 3.5 times the baseline group mean R-value.Individual R-values for Group 2 monkeys are shown in Table 28; theindividual R-values are also plotted against the time points of theblood sample in FIG. 131. All monkeys in this group showed aprolongation of their R-values in response to administration of FVIII Abcompared to their baseline pre-treatment R-values at baseline (range 4.0to 9.1-fold). All but one of the monkeys in this group also exhibited areduction of their R-values in response to administration of 1 mg/kgARC19499 compared to their baseline pre-treatment R-values (range 2.4 to5.0-fold); treatment of these monkeys with two doses of 1 mg/kgARC19499, which did correct all of the bleed times in this group,reduced further the R-values in two of these monkeys, and slightlyincreased the R-value in the third monkey. One monkey, NHPO611655,showed a 103% increase in the R-value compared with the R-value postFVIII Ab injection after administration of 1 mg/kg ARC19499; treatmentof this monkey with an additional 1 mg/kg ARC19499, which did correctthe bleeding time, produced a decrease in the R-value to 6.4 times thebaseline R-value.

TABLE 27 Mean Whole Blood TEG ® R-values in Monkeys Whose Bleeding Timeswere Corrected by Two Doses of 1 mg/kg ARC19499 (Group 2) Group 2: MeanWhole Blood TEG ® R-values Time point Mean SEM (min) (min) (min)Baseline −10 6.0 1.0 2 hr post FVIII ab 120 36 4.5 treatment 55 min postfirst 210 24 8.7 ARC19499 treatment 12 min post second 237 21 7.1ARC19499 treatment

TABLE 28 Individual Whole Blood TEG ® R-values in Monkeys Whose BleedingTimes were Corrected by Two Doses of 1 mg/kg ARC19499 (Group 2) Group 2:Individual Whole Blood TEG ® R-values Time NHP NHP NHP NHP point 06103010702277 0611655 0511011 (min) (min) (min) (min) (min) Baseline −10 ns8.1 6.0 4.0 2 hr post FVIII ab 120 46.3 35.8 24.1 36.2 treatment 55 minpost first 210 8.2 19.8 48.9 19.9 ARC19499 treatment 12 min post 237 6.711.9 38.3 25.6 ARC19499 treatment

The R-values for the Group 3 monkey are shown in Table 29; the R-valuesare also plotted against the time points of the blood samples in FIG.132. This monkey showed, in response to administration of FVIII Ab, aprolongation of its R-value to 7.6 times its baseline R-value. Inresponse to treatment with a 1 mg/kg dose of ARC19499, which did notcorrect the bleed time, the R-value was reduced to 6.1-fold of thebaseline R-value. Treatment of this monkey with two doses of 1 mg/kgARC19499, which also did not correct the bleed time in this monkey,further reduced the R-value slightly, to 5.8 times that of the baseline.An additional dose of 1 mg/kg ARC19499, which did correct the bleedtime, reduced the R-value to 3 times the baseline R-value.

TABLE 29 Whole Blood TEG ® R-values in a Monkey Whose Bleeding Time wasCorrected by Three Doses of 1 mg/kg ARC19499 (Group 3) Group 3: WholeBlood TEG ® R-values Time point NHP0702073 (min) (min) Baseline −10 3.92 hr post FVIII ab 120 29.5 treatment 55 min post first 210 23.6ARC19499 treatment 12 min post second 239 22.5 ARC19499 treatment 12 minpost third 278 11.8 ARC19499 treatment

The R-values for the Group 4 monkey are shown in Table 30; the R-valuesare also plotted against the time points of the blood samples in FIG.133. This monkey showed, in response to administration of FVIII Ab, aprolongation to 2.9-fold times its baseline R-value. In response totreatment with 1 mg/kg dose of ARC19499, which did not correct the bleedtime, the R-value was reduced to 1.6 fold times the baseline R-value.Treatment of this monkey with two doses of 1 mg/kg ARC19499, which alsodid not correct the bleed time in this monkey, further reduced theR-value slightly, to 1.5-fold times the baseline R-value. An additionaldose of 1 mg/kg ARC19499, which still did not correct the bleed time,reduced the R-value slightly more to 1.2 times the baseline R-value. Afourth dose of 3 mg/kg ARC19499 had little effect on the R-value, whichwas now 1.1 times the pre-treatment baseline R-value. No additionalbleeding time assessments could be done on this animal due to a lacksufficient available vein consistent with that used for previousassessments.

TABLE 30 Whole Blood TEG ® R-values in a Monkey Whose Bleeding Time wasNot Corrected by Three Doses of 1 mg/kg ARC19499 (Group 4) Group 4:Whole Blood TEG ® R-values Time point NHP0607367 (min) (min) Baseline−10 7.2 2 hr post FVIII ab 120 21.2 treatment 55 min post first 210 11.2ARC19499 treatment 12 min post second 237 10.7 ARC19499 treatment 12 minpost third 264 8.8 ARC19499 treatment 12 min post fourth 296 8.1ARC19499 treatment

The coagulation status of the cynomolgus plasma was also analyzed usingthe TEG® assay on plasma from the citrated blood samples. Citrated wholeblood samples were kept at room temperature until plasma processing.Samples were centrifuged at 2,000×g for 15 minutes at room temperature.The plasma was removed and immediately stored at −80° C. until shippedfor analysis. Prior to analysis, plasma samples were thawed rapidly at37° C. To initiate the clotting reaction, 330 μL citrated plasma wasadded to a disposable cup (Haemonetics Corporation, Braintree, Mass.,cat no. 6211) containing 20 μL 0.2 M (Haemonetics Corporation(Braintree, Mass.)) and 10 μL TF (final dilution of 1:200000 at 37° C.)Innovin (Dade-Behring, Newark, Del.) was used as a tissue factor (TF)source, reconstituted in water as per manufacturer recommendation, anddiluted 1:5555 in 0.9% saline prior to use. Reconstituted stock Innovinwas stored at 4° C. for less than 4 weeks. The time to initial clotformation (R-value) was measured using the Haemoscope TEG® 5000 system(Haemonetics Corporation, Braintree, Mass.).

The mean group R-values (±SEM) of plasma samples from Group 1 monkeys(whose bleeding times were corrected with one dose of 1 mg/kg ARC19499)are shown in Table 31. The mean group R-values are also plotted againstthe time points of the plasma sample (FIG. 134). Treatment with FVIII Abresulted in a prolongation of the group mean R-value to about 2.8 timesthe group mean R-value at baseline. While treatment of the monkeys with1 mg/kg ARC19499 reduced the group mean R-value from that obtained afterFVIII Ab treatment, this R-value was still about 1.9 times the groupmean baseline R-value. Individual R-values for Group 1 monkeys are shownin Table 32; the individual R-values are also plotted against the timepoints of the plasma sample in FIG. 135. All monkeys in this groupshowed a prolongation of their R-values in response to administration ofFVIII Ab compared to their baseline pre-treatment R-values (range 1.7 to5.1-fold). All but one of the monkeys in this group also exhibited areduction of their R-values in response to administration of 1 mg/kgARC19499 compared to their baseline pre-treatment R-values (range 1.3 to1.8-fold). One monkey, NHP0603477, showed a 50% increase in the R-valuecompared with the R-value post FVIII Ab injection after administrationof 1 mg/kg ARC19499; the whole blood TEG® analysis had instead shown a53% decrease in the R-value compared with the R-value post FVIII Abinjection after administration of 1 mg/kg ARC19499. The plasma TEG®analysis of NHP0701565, whose whole blood TEG® analysis had shown aslight increase in R-value in response to administration of 1 mg/kgARC19499, was actually 66% below the R-value post FVIII Ab injectionafter administration of 1 mg/kg ARC19499.

TABLE 31 Mean Plasma TEG ® R-values in Monkeys Whose Bleeding Times wereCorrected by one Dose of 1 mg/kg ARC19499 (Group 1) Group 1: Mean PlasmaTEG ® R-values Time point Mean SEM (min) (min) (min) Baseline −10 5.40.37 2 hr post FVIII ab 120 15 1.6 treatment 55 min post 210 10 1.7ARC19499 treatment

TABLE 32 Individual Plasma TEG ® R-values in Monkeys Whose BleedingTimes were Corrected by One Dose of 1 mg/kg ARC19499 (Group 1) Group 1:Individual Plasma TEG ® R-values Time NHP NHP NHP NHP NHP NHP point0703333 0704039 0701565 0604313 0703551 0603477 (min) (min) (min) (min)(min) (min) (min) Baseline −10 5.0 6.0 4.3 4.6 6.3 6.4 2 hr post FVIIIab 120 12.2 16.6 21.9 15.5  14.1  10.8 treatment 55 min post 210 9.2 7.87.4 ns ns 16.2 ARC19499 treatment

The mean group R-values (±SEM) of plasma samples from Group 2 monkeys(whose bleeding times were corrected with two doses of 1 mg/kg ARC19499)are shown in Table 33. The mean group R-values are also plotted againstthe time points of the blood samples in FIG. 136. Treatment with theanti-FVIII antibody resulted in a prolongation of the group mean R-valueto about 4.0-fold of the group mean R-value at baseline. In response totreatment with 1 mg/kg dose of ARC19499, which did not correct the bleedtime, the group mean R-value was reduced to about 2.2-fold times thebaseline group mean R-value. Treatment of the monkeys with two doses of1 mg/kg ARC19499, which did correct the bleed time in this group, didnot significantly reduce further the group mean R-value from thatobtained after FVIII Ab treatment. Individual R-values for Group 2monkeys are shown in Table 34; the individual R-values are also plottedagainst the time points of the blood sample in FIG. 137. All monkeys inthis group showed a prolongation of their R-values in response toadministration of FVIII Ab compared to their baseline pre-treatmentR-values at baseline (range 1.1 to 6.4-fold). In response to treatmentwith 1 mg/kg dose of ARC19499, which did not correct the bleed times,the plasma R-values of all monkeys in this group were reduced over thebaseline group mean R-value (range 0.5 to 4.5-fold). Treatment of themonkeys with two doses of 1 mg/kg ARC19499, which did correct all of thebleed times in this group, produced unchanged or further reducedR-values, although in two monkeys the R-values were still higher thanthose at the pre-treatment baseline (range 0.5 to 3.2-fold).

TABLE 33 Mean Plasma TEG ® R-values in Monkeys Whose Bleeding Times wereCorrected by Two Doses of 1 mg/kg ARC19499 (Group 2) Group 2: MeanPlasma TEG ® R-values Time point Mean SEM (min) (min) (min) Baseline −105.8 0.6 2 hr post FVIII ab 120 23 6.5 treatment 55 min post first 210 134.5 ARC19499 treatment 12 min post second 237 12 3.7 ARC19499 treatment

TABLE 34 Individual Plasma TEG ® R-values in Monkeys Whose BleedingTimes were Corrected by Two Doses of 1 mg/kg ARC19499 (Group 2) Group 2:Individual Plasma TEG ® R-values Time NHP NHP NHP NHP point 06103010702277 0611655 0511011 (min) (min) (min) (min) (min) Baseline −10 4.67.2 5.3 6.0 2 hr post FVIII ab 120 15.4 8.1 33.9 33.7 treatment 55 minpost first 210 7.6 3.5 23.7 16.8 ARC19499 treatment 12 min post 237 ns3.9 17.2 16.5 ARC19499 treatment

The R-values of the plasma samples from the Group 3 monkey are shown inTable 35; the R-values are also plotted against the time points of theblood samples in FIG. 138. This monkey showed, in response toadministration of FVIII Ab, a prolongation of its R-value to 3.5 foldtimes its baseline R-value. In response to treatment with 1 mg/kg doseof ARC19499, which did not correct the bleed time, the R-value wasreduced to 1.3 times the baseline R-value. Treatment of this monkey withtwo doses of 1 mg/kg ARC19499, which also did not correct the bleed timein this monkey, further reduced the R-value to 1.1 times that of thebaseline. An additional dose of 1 mg/kg ARC19499, which did correct thebleed time, reduced the R-value to 0.9 times the baseline R-value.

TABLE 35 Plasma TEG ® R-values in a Monkey Whose Bleeding Time wasCorrected by Three Doses of 1 mg/kg ARC19499 (Group 3) Group 3: PlasmaTEG ® R-values Time point NHP0702073 (min) (min) Baseline −10 4.7 2 hrpost FVIII ab 120 16.4 treatment 55 min post first 210 6.2 ARC19499treatment 12 min post second 239 5.2 ARC19499 treatment 12 min postthird 278 4.4 ARC19499 treatment

The R-values of plasma samples from the Group 4 monkey are shown inTable 36; the R-values are also plotted against the time points of theblood samples in FIG. 139. This monkey showed, in response toadministration of FVIII Ab, a prolongation to 2.9 times its baselineR-value. In response to treatment with 1 mg/kg dose of ARC19499, whichdid not correct the bleed time, the R-value was reduced over thebaseline R-value to 1.1 times the baseline R-value. Plasma from bloodtaken after treatment of this monkey with two doses of 1 mg/kg ARC19499,which also did not correct the bleed time in this monkey, exhibited aslightly higher R-value of 1.2 times the baseline R-value. An additionaldose of 1 mg/kg ARC19499, which still did not correct the bleed time,reduced the R-value slightly more to 1.0 times the baseline R-value. Afourth dose of 3 mg/kg ARC19499 reduced the R-value to below thepre-treatment baseline (0.9 times the pre-treatment baseline R-value).No additional bleeding time assessments could be done on this animal dueto a lack sufficient available vein consistent with that used forprevious assessments.

TABLE 36 Plasma TEG ® R-values in a Monkey Whose Bleeding Time was NotCorrected by Three Doses of 1 mg/kg ARC19499 (Group 4) Group 4: PlasmaTEG ® R-values Time point NHP0607367 (min) (min) Baseline −10 6.4 2 hrpost FVIII ab 120 18.5 treatment 55 min post first 210 6.8 ARC19499treatment 12 min post second 237 7.5 ARC19499 treatment 12 min postthird 264 6.2 ARC19499 treatment 12 min post fourth 296 5.8 ARC19499treatment

The above example shows that monkeys treated with the FVIII Ab exhibitedprolonged bleeding after puncture of the saphenous vein, an observationconsistent with the prolonged bleeding that is the hallmark ofhemophilia. In a majority of monkeys tested in this study (11 of 12),treatment with up to 3 mg/kg ARC19499 corrected this prolonged bleedingtime. Six of the monkeys only required one dose of 1 mg/kg ARC19499 toexhibit this correction; bleeding time in four other monkeys wascorrected with two doses of 1 mg/kg ARC19499. R-values in TEG® analysisof the whole blood from these monkeys exhibited the expected elevationafter FVIII Ab administration; this elevation was reduced towardsbaseline levels after treatment with ARC19499; a similar pattern wasseen in the analysis of the plasma from these blood samples. These datashow that ARC19499 can correct prolonged bleeding in a model of inducedhemophilia, supporting the potential clinical utility of ARC19499 as asuccessful therapeutic in the treatment of inhibitor and non-inhibitorhemophilia A patients.

Example 34

This example is an evaluation of tolerated and non-toleratedsubstitutions in ARC17480 through aptamer medicinal chemistry.

Molecules were generated for testing with modifications at the2′-position or within the phosphate backbone of residues in ARC17480, asshown in Table 37 below and in FIGS. 140 and 141. Each individual2′-deoxy residue in ARC17480 was replaced by the corresponding2′-methoxy or 2′-fluoro containing residue, resulting inARC18538-ARC18541 and ARC19493-ARC19496, respectively (FIG. 140).Additionally, some molecules with multiple 2′-deoxy to 2′-methoxy and/or2′-deoxy to 2′-fluoro residues were generated at the four deoxycytidineresidues in ARC17480 at positions 9, 14, 16 and 25, resulting inARC18545, ARC18546, ARC18549, ARC19476, ARC19477, ARC19478, ARC19484,ARC19490 and ARC19491 (FIG. 140). Each individual 2′-methoxy residue inARC17480 was replaced by the corresponding 2′-deoxy residue, with2′-methoxyuridine residues replaced with both 2′-deoxythymidine and2′-deoxyuridine residues, resulting in ARC19448-ARC19475 andARC33867-ARC33877 (FIG. 140). The phosphate between each pair ofnucleotides in ARC17480 was replaced individually with aphosphorothioate, resulting in ARC19416-ARC19447 (FIG. 141).

The modified ARC17480 molecules were assayed for binding and function.The assays used for this evaluation were the calibrated automatedthrombogram (CAT) assay, a dot-blot binding-competition assay and a FXaactivity assay. The results of these assays are summarized in Table 37and depicted in FIG. 142. A substitution was considered tolerated foractivity if it met the criteria of at least two of the three assays thatwere carried out. Substitutions that were tolerated (“yes”) and nottolerated (“no”) are identified in Table 37. The experimental detailsand the criteria used for each assay are described in the followingparagraphs.

The TFPI-inhibitory activity of each molecule was evaluated in the CATassay in pooled hemophilia A plasma at 500 nM, 166.67 nM, 55.56 nM,18.52 nM, 6.17 nM and 2.08 nM aptamer concentration. ARC17480 wasincluded in every experiment as a control. For each molecule, theendogenous thrombin potential (ETP) and peak thrombin values at eachaptamer concentration were used for analysis. The ETP or peak thrombinvalue for hemophilia A plasma alone was subtracted from thecorresponding value in the presence of aptamer for each molecule at eachconcentration. Then, the corrected ETP and peak values were plotted as afunction of aptamer concentration and fit to the equationy=(max/(1+IC₅₀/x))+int, where y=ETP or peak thrombin, x=concentration ofaptamer, max=the maximum ETP or peak thrombin, and int=the y-intercept,to generate an IC₅₀ value for both the ETP and the peak thrombin. TheIC₅₀ of each aptamer was compared to the IC₅₀ of ARC17480 that wasevaluated in the same experiment. A substitution was consideredtolerated in the CAT assay if both the ETP and peak thrombin IC_(so) ofthat molecule were not more than 5-fold greater than that of ARC17480evaluated in the same experiment. Tolerated substitutions are indicatedin Table 37 as meeting the assay criteria (“yes”) or not meeting theassay criteria (“no”).

Each molecule was evaluated for binding to tissue factor pathwayinhibitor (TFPI) in a binding-competition assay. For these experiments,10 nM human TFPI (American Diagnostica, Stamford, Conn., catalog#4500PC) was incubated with trace amounts of radiolabeled ARC17480 and5000 nM, 1666.67 nM, 555.56 nM, 185.19 nM, 61.73 nM, 20.58 nM, 6.86 nM,2.29 nM, 0.76 nM or 0.25 nM of unlabeled competitor aptamer. ARC17480was included as a competitor in every experiment as a control. For eachmolecule, the percentage of radiolabeled ARC17480 bound at eachcompetitor aptamer concentration was used for analysis. The percentageof radiolabeled ARC17480 bound was plotted as a function of aptamerconcentration and fit to the equation y=(max/(1+x/IC₅₀))+int, wherey=the percentage of radiolabeled ARC17480 bound, x=the concentration ofaptamer, max=the maximum radiolabeled ARC17480 bound, and int=they-intercept, to generate an IC₅₀ value for binding-competition. The IC₅₀of each aptamer was compared to the IC₅₀ of ARC17480 that was evaluatedin the same experiment. A substitution was considered tolerated in thebinding-competition assay if the IC_(so) of that molecule was not morethan 5-fold greater than that of ARC17480 evaluated in the sameexperiment. Tolerated substitutions are indicated in Table 37 as meetingthe assay criteria (“yes”) or not meeting the assay criteria (“no”).

Each molecule was evaluated for inhibition of TFPI in a Factor Xa (FXa)activity assay. The ability of FXa to cleave a chromogenic substrate wasmeasured in the presence and absence of TFPI, with or without theaddition of aptamer. For these experiments, 2 nM human FXa was incubatedwith 8 nM human TFPI. Then, 500 μIM chromogenic substrate and aptamerswere added and FXa cleavage of the substrate was measured by absorbanceat 405 nm (A₄₀₅) as a function of time. Aptamers were tested at 500 nM,125 nM, 31.25 nM, 7.81 nM, 1.95 nM and 0.49 nM concentrations. ARC17480was included as a control in each experiment. For each aptamerconcentration, the A₄₀₅ was plotted as a function of time and the linearregion of each curve was fit to the equation y=mx+b, where y=A₄₀₅, x=theaptamer concentration, m=the rate of substrate cleavage, and b=they-intercept, to generate a rate of FXa substrate cleavage. The rate ofFXa substrate cleavage in the presence of TFPI and the absence ofaptamer was subtracted from the corresponding value in the presence ofboth TFPI and aptamer for each molecule at each concentration. Then, thecorrected rates were plotted as a function of aptamer concentration andfit to the equation y=(V_(max)/(1+IC₅₀/x)), where y=the rate ofsubstrate cleavage, x=concentration of aptamer, and V_(max)=the maximumrate of substrate cleavage, to generate an IC₅₀ and maximum (V_(max))value. The IC₅₀ and V_(max) values of each aptamer were compared to theIC₅₀ and V_(max) values of ARC17480 that was evaluated in the sameexperiment. A substitution was considered tolerated in the FXa activityassay if the IC₅₀ of that molecule was not more than 5-fold greater thanthat of ARC17480 evaluated in the same experiment and the V_(max) valuewas not less than 80% of the V_(max) value of the ARC17480 evaluated inthe same experiment. Tolerated substitutions are indicated in Table 37as meeting the assay criteria (“yes”) or not meeting the assay criteria(“no”).

This example demonstrates that multiple individual 2′-substitutions inARC17480 are tolerated for binding and activity, and that somecombinations of 2′-substitutions are also tolerated (Table 37 and FIG.142). This example also demonstrates that a phosphorothioatesubstitution is tolerated between each pair of nucleotides in ARC17480(Table 37). Additional combinations of tolerated 2′-substitutions and/orphosphorothioate substitutions in ARC17480 will likely be tolerated forbinding and activity.

TABLE 37 Tolerated and Non-tolerated Substitutions in ARC17480Sequence (5′ → 3′) (3T = inverted dT; s = phosphorothioate; mN = 2′-Meets Criteria? SEQ methoxy-containing residue; Binding- FXa Substi- IDARC dN = deoxy residue; fN = 2′- CAT competition Activity tution(s) NO:# fluoro-containing residue) Assay Assay Assay Tolerated 19 18538mGmGmAmAmUmAmUmAmCmU NO NO NO NO mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 20 18539 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGmCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 21 18540mGmGmAmAmUmAmUmAdCmU YES YES NO YES mUmGmGdCmUmCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 22 18541 mGmGmAmAmUmAmUmAdCmU YES YES NO YESmUmGmGdCmUdCmGmUmUmA mGmGmUmGmCmGmUmAmUmA mUmA3T 23 19493mGmGmAmAmUmAmUmAfCmUm NO NO NO NO UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 24 19494 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGfCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 25 19495mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUfCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 26 19496 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGdCmUdCmGmUmUmA mGmGmUmGfCmGmUmAmUmA mUmA3T 27 19448dGmGmAmAmUmAmUmAdCmUm YES YES YES YES UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 28 19449 mGdGmAmAmUmAmUmAdCmUm YES YES YESYES UmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 29 19450mGmGdAmAmUmAmUmAdCmUm YES YES YES YES UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 30 19451 mGmGmAdAmUmAmUmAdCmUm YES YES YESYES UmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 31 19452mGmGmAmAdTmAmUmAdCmUm YES YES YES YES UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 32 19453 mGmGmAmAmUdAmUmAdCmUm YES YES YESYES UmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 33 19454mGmGmAmAmUmAdTmAdCmUm YES YES YES YES UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 34 19455 mGmGmAmAmUmAmUdAdCmUm YES YES YESYES UmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 35 19456mGmGmAmAmUmAmUmAdCdTm NO YES NO NO UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 36 19457 mGmGmAmAmUmAmUmAdCmUd YES YES YESYES TmGmGdCmUdCmGmUmUmAmG mGmUmGdCmGmUmAmUmAmU mA3T 37 19458mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUdGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 38 19459 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGdGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 39 19460mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCdTdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 40 19461 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGdCmUdCdGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 41 19462mGmGmAmAmUmAmUmAdCmU NO YES NO NO mUmGmGdCmUdCmGdTmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 42 19463 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGdCmUdCmGmUdTmAm GmGmUmGdCmGmUmAmUmAm UmA3T 43 19464mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUdAmGmGmUmGdCmGmUmAmUmAm UmA3T 44 19465 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGdCmUdCmGmUmUmAd GmGmUmGdCmGmUmAmUmAm UmA3T 45 19466mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGdGmUmGdCmGmUmAmUmA mUmA3T 46 19467 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGdTmGdCmGmUmAmUmA mUmA3T 47 19468mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUdGdCmGmUmAmUmA mUmA3T 48 19469 mGmGmAmAmUmAmUmAdCmU NO NO YES NOmUmGmGdCmUdCmGmUmUmA mGmGmUmGdCdGmUmAmUmA mUmA3T 49 19470mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGdTmAmUmA mUmA3T 50 19471 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUdAmUmA mUmA3T 51 19472mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAdTmA mUmA3T 52 19473 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUdA mUmA3T 53 19474mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA dTmA3T 54 19475 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUdA3T 55 33867mGmGmAmAdUmAmUmAdCmUm YES YES YES YES UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 56 33868 mGmGmAmAmUmAdUmAdCmUm YES YES YESYES UmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 57 33869mGmGmAmAmUmAmUmAdCdUm NO NO NO NO UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 58 33870 mGmGmAmAmUmAmUmAdCmUd YES NO YES YESUmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 59 33871mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCdUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 60 33872 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGdCmUdCmGdUmUmAm GmGmUmGdCmGmUmAmUmAm UmA3T 61 33873mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUdUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 62 33874 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGdCmUdCmGmUmUmA mGmGdUmGdCmGmUmAmUmA mUmA3T 63 33875mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGdUmAmUmA mUmA3T 64 33876 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAdUmA mUmA3T 65 33877mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA dUmA3T 66 18545 mGmGmAmAmUmAmUmAdCmU YES YES NO YESmUmGmGmCmUmCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 8 18546mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGmCmUdCmGmUmUmAmGmGmUmGmCmGmUmAmUmA mUmA3T 67 18549 mGmGmAmAmUmAmUmAdCmU YES YES NO YESmUmGmGmCmUmCmGmUmUmA mGmGmUmGmCmGmUmAmUmA mUmA3T 68 19476mGmGmAmAmUmAmUmAfCmUm NO YES YES YES UmGmGmCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T 69 19477 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGmCmUfCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 70 19478mGmGmAmAmUmAmUmAdCmU NO YES YES YES mUmGmGmCmUdCmGmUmUmAmGmGmUmGfCmGmUmAmUmA mUmA3T 71 19484 mGmGmAmAmUmAmUmAdCmU YES NO YES YESmUmGmGfCmUdCmGmUmUmAm GmGmUmGmCmGmUmAmUmAm UmA3T 72 19490mGmGmAmAmUmAmUmAfCmUm YES YES NO YES UmGmGmCmUdCmGmUmUmAmGmGmUmGmCmGmUmAmUmAm UmA3T 73 19491 mGmGmAmAmUmAmUmAdCmU YES YES YES YESmUmGmGmCmUfCmGmUmUmA mGmGmUmGmCmGmUmAmUmA mUmA3T 74 19416mGsmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 75 19417 mGmGsmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 76 19418mGmGmAsmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 77 19419 mGmGmAmAsmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 78 19420mGmGmAmAmUsmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 79 19421 mGmGmAmAmUmAsmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 80 19422mGmGmAmAmUmAmUsmAdCmU YES YES NO YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 81 19423 mGmGmAmAmUmAmUmAsdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 82 19424mGmGmAmAmUmAmUmAdCsmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 83 19425 mGmGmAmAmUmAmUmAdCmUs YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 84 19426mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUsmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 85 19427 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGsmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 86 19428mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGsdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 87 19429 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCsmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 88 19430mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUsdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 89 19431 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCsmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 90 19432mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGsmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 91 19433 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUsmUmA mGmGmUmGdCmGmUmAmUmA mUmA3T 92 19434mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUsmAmGmGmUmGdCmGmUmAmUmA mUmA3T 93 19435 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmAs mGmGmUmGdCmGmUmAmUmA mUmA3T 94 19436mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGsmGmUmGdCmGmUmAmUm AmUmA3T 95 19437 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGsmUmGdCmGmUmAmUm AmUmA3T 96 19438mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUsmGdCmGmUmAmUm AmUmA3T 97 19439 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGsdCmGmUmAmUm AmUmA3T 98 19440mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCsmGmUmAmUm AmUmA3T 99 19441 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGsmUmAmUm AmUmA3T 100 19442mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUsmAmUm AmUmA3T 101 19443 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAsmUm AmUmA3T 102 19444mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUsm AmUmA3T 103 19445 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA smUmA3T 104 19446mGmGmAmAmUmAmUmAdCmU YES YES YES YES mUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUsmA3T 105 19447 mGmGmAmAmUmAmUmAdCmU YES YES YESYES mUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA mUmAs3T

Example 35

This example is an evaluation of tolerated and non-tolerated deletionsin ARC17480.

Molecules were generated for testing with single or multiple residuesdeleted in the ARC17480 sequence, as shown in Table 38 below and in FIG.143. Each individual residue in ARC17480 was deleted one at a time,resulting in ARC32301, ARC33120-ARC33143, and ARC18555. In cases wheretwo adjacent nucleotides were identical, the corresponding doubledeletion was also generated, resulting in ARC32302 andARC33144-ARC33148. Additionally, molecules with multiple deletions weregenerated, resulting in ARC32303, ARC32305, ARC32306, ARC32307,ARC33889, ARC33890, ARC33891, ARC33895, ARC33900 and ARC33907.

The ARC17480 molecules with deletions were assayed for binding andfunction. The assays used for this evaluation were the calibratedautomated thrombogram (CAT) assay, a dot-blot binding-competition assayand a FXa activity assay. The results of these assays are summarized inTable 38 and FIG. 144. A substitution was considered tolerated foractivity if it met the criteria of at least two of the three assays thatwere carried out. Substitutions that were tolerated (“yes”) and nottolerated (“no”) are identified in Table 38. The experimental detailsand the criteria used for each assay are described in the followingparagraphs.

The TFPI-inhibitory activity of each molecule was evaluated in the CATassay in pooled hemophilia A plasma at 500 nM, 166.67 nM, 55.56 nM,18.52 nM, 6.17 nM and 2.08 nM aptamer concentration. ARC17480 wasincluded in every experiment as a control. For each molecule, theendogenous thrombin potential (ETP) and peak thrombin values at eachaptamer concentration were used for analysis. The ETP or peak thrombinvalue for hemophilia A plasma alone was subtracted from thecorresponding value in the presence of aptamer for each molecule at eachconcentration. Then, the corrected ETP and peak values were plotted as afunction of aptamer concentration and fit to the equationy=(max/(1+IC₅₀/x))+int, where y=ETP or peak thrombin, x=concentration ofaptamer, max=the maximum ETP or peak thrombin, and int=the y-intercept,to generate an IC₅₀ value for both the ETP and the peak thrombin. TheIC₅₀ of each aptamer was compared to the IC₅₀ of ARC17480 that wasevaluated in the same experiment. A substitution was consideredtolerated in the CAT assay if both the ETP and peak thrombin IC_(so) ofthat molecule were not more than 5-fold greater than that of ARC17480evaluated in the same experiment. Tolerated substitutions are indicatedin Table 38 as meeting the assay criteria (“yes”) or not meeting theassay criteria (“no”).

Each molecule was evaluated for binding to tissue factor pathwayinhibitor (TFPI) in a binding-competition assay. For these experiments,10 nM human TFPI (American Diagnostica, Stamford, Conn., catalog#4500PC) was incubated with trace amounts of radiolabeled ARC17480 and5000 nM, 1666.67 nM, 555.56 nM, 185.19 nM, 61.73 nM, 20.58 nM, 6.86 nM,2.29 nM, 0.76 nM or 0.25 nM of unlabeled competitor aptamer. ARC17480was included as a competitor in every experiment as a control. For eachmolecule, the percentage of radiolabeled ARC17480 bound at eachcompetitor aptamer concentration was used for analysis. The percentageof radiolabeled ARC17480 bound was plotted as a function of aptamerconcentration and fit to the equation y=(max/(1+x/IC₅₀))+int, wherey=the percentage of radiolabeled ARC17480 bound, x=the concentration ofaptamer, max=the maximum radiolabeled ARC17480 bound, and int=they-intercept, to generate an IC₅₀ value for binding-competition. The IC₅₀of each aptamer was compared to the IC₅₀ of ARC17480 that was evaluatedin the same experiment. A substitution was considered tolerated in thebinding-competition assay if the IC_(so) of that molecule was not morethan 5-fold greater than that of ARC17480 evaluated in the sameexperiment. Tolerated substitutions are indicated in Table 38 as meetingthe assay criteria (“yes”) or not meeting the assay criteria (“no”).

Each molecule was evaluated for inhibition of TFPI in a Factor Xa (FXa)activity assay. The ability of FXa to cleave a chromogenic substrate wasmeasured in the presence and absence of TFPI, with or without theaddition of aptamer. For these experiments, 2 nM human FXa was incubatedwith 8 nM human TFPI. Then, 500 μM chromogenic substrate and aptamerswere added and FXa cleavage of the substrate was measured by absorbanceat 405 nm (A₄₀₅) as a function of time. Aptamers were tested at 500 nM,125 nM, 31.25 nM, 7.81 nM, 1.95 nM and 0.49 nM concentrations. ARC17480was included as a control in each experiment. For each aptamerconcentration, the A₄₀₅ was plotted as a function of time and the linearregion of each curve was fit to the equation y=mx+b, where y=A₄₀₅, x=theaptamer concentration, m=the rate of substrate cleavage, and b=they-intercept, to generate a rate of FXa substrate cleavage. The rate ofFXa substrate cleavage in the presence of TFPI and the absence ofaptamer was subtracted from the corresponding value in the presence ofboth TFPI and aptamer for each molecule at each concentration. Then, thecorrected rates were plotted as a function of aptamer concentration andfit to the equation y=(V_(max)/(1+IC₅₀/x)), where y=the rate ofsubstrate cleavage, x=concentration of aptamer, and V_(max)=the maximumrate of substrate cleavage, to generate an IC₅₀ and maximum (V_(max))value. The IC₅₀ and V_(max) values of each aptamer were compared to theIC₅₀ and max values of ARC17480 that was evaluated in the sameexperiment. A substitution was considered tolerated in the FXa activityassay if the IC₅₀ of that molecule was not more than 5-fold greater thanthat of ARC17480 evaluated in the same experiment and the V_(max) valuewas not less than 80% of the V_(max) value of the ARC17480 evaluated inthe same experiment. Tolerated substitutions are indicated in Table 38as meeting the assay criteria (“yes”) or not meeting the assay criteria(“no”).

This example demonstrates that multiple individual deletions in ARC17480are tolerated for binding and activity, and that some combinations ofdeletions are also tolerated (Table 38 and FIG. 144). ARC33889 andARC33895 each tolerate a total of seven deletions at their 5′- and3′-ends, resulting in core molecules that are twenty-five nucleotideslong. Additional combinations of deletions in ARC17480 may be toleratedfor binding and activity, with or without additional changes in themolecule.

TABLE 38 Tolerated and Non-tolerated Deletions in ARC17480 Sequence (5′→ 3′) Meets Criteria? SEQ (3T = inverted dT; mN = 2′- Binding- FXa/Substi- ID ARC methoxy-containing residue; CAT competition Activitytution(s) NO: # dN = deoxy residue) Assay Assay Assay Tolerated 10632301 mGmAmAmUmAmUmAdCmUmU YES YES YES YES mGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 107 33120 mGmGmAmUmAmUmAdCmUm YES YES YES YESUmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmA mUmA3T 108 33121mGmGmAmAmAmUmAdCmUm YES YES YES YES UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 109 33122 mGmGmAmAmUmUmAdCmUm NO YES YES YESUmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmA mUmA3T 110 33123mGmGmAmAmUmAmAdCmUm NO NO NO NO UmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAmUmA3T 111 33124 mGmGmAmAmUmAmUdCmUm NO NO NO NO UmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 112 33125 mGmGmAmAmUmAmUmAmUm NO NO NO NOUmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmA mUmA3T 113 33126mGmGmAmAmUmAmUmAdCm NO NO NO NO UmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAmUmA3T 114 33127 mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 115 33128 mGmGmAmAmUmAmUmAdCm NO NO NO NOUmUmGmGmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUm AmUmA3T 116 33129mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGmGdCdCmGmUmUmAm GmGmUmGdCmGmUmAmUmAmUmA3T 117 33130 mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGmGdCmUmGmUmUmAmGmGmUmGdCmGmUmAmUm AmUmA3T 118 33131 mGmGmAmAmUmAmUmAdCm NO NO NO NOUmUmGmGdCmUdCmUmUmAm GmGmUmGdCmGmUmAmUmA mUmA3T 119 33132mGmGmAmAmUmAmUmAdCm NO NO YES NO UmUmGmGdCmUdCmGmUmAmGmGmUmGdCmGmUmAmUmA mUmA3T 120 33133 mGmGmAmAmUmAmUmAdCm NO NO NO NOUmUmGmGdCmUdCmGmUmU mGmGmUmGdCmGmUmAmUm AmUmA3T 121 33134mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGmGdCmUdCmGmUmU mAmGmUmGdCmGmUmAmUmAmUmA3T 122 33135 mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGmGdCmUdCmGmUmUmAmGmGmGdCmGmUmAmUm AmUmA3T 123 33136 mGmGmAmAmUmAmUmAdCm NO NO NO NOUmUmGmGdCmUdCmGmUmU mAmGmGmUdCmGmUmAmUm AmUmA3T 124 33137mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGmGdCmUdCmGmUmU mAmGmGmUmGmGmUmAmUmAmUmA3T 125 33138 mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmUmAmUm AmUmA3T 126 33139 mGmGmAmAmUmAmUmAdCm NO NO NO NOUmUmGmGdCmUdCmGmUmU mAmGmGmUmGdCmGmAmUm AmUmA3T 127 33140mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGmGdCmUdCmGmUmU mAmGmGmUmGdCmGmUmUmAmUmA3T 128 33141 mGmGmAmAmUmAmUmAdCm NO YES YES YES UmUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAm AmUmA3T 129 33142 mGmGmAmAmUmAmUmAdCm YES YES YESYES UmUmGmGdCmUdCmGmUmU mAmGmGmUmGdCmGmUmAm UmUmA3T 130 33143mGmGmAmAmUmAmUmAdCm YES YES YES YES UmUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAm UmAmA3T 131 18555 mGmGmAmAmUmAmUmAdCm YES YES YESYES UmUmGmGdCmUdCmGmUmU mAmGmGmUmGdCmGmUmAm UmAmU3T 132 32302mAmAmUmAmUmAdCmUmUmG YES YES YES YES mGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAmU mA3T 133 33144 mGmGmUmAmUmAdCmUmUm YES YES YES YESGmGdCmUdCmGmUmUmAmG mGmUmGdCmGmUmAmUmAm UmA3T 134 33145mGmGmAmAmUmAmUmAdCm NO NO NO NO GmGdCmUdCmGmUmUmAmG mGmUmGdCmGmUmAmUmAmUmA3T 135 33146 mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAmU mA3T 136 33147 mGmGmAmAmUmAmUmAdCm NO NO NO NOUmUmGmGdCmUdCmGmAmG mGmUmGdCmGmUmAmUmAm UmA3T 137 33148mGmGmAmAmUmAmUmAdCm NO NO NO NO UmUmGmGdCmUdCmGmUmU mAmUmGdCmGmUmAmUmAmUmA3T 138 32303 mAmUmAmUmAdCmUmUmGm YES YES YES YES GdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAmUm A3T 139 32305 mGmAmAmUmAmUmAdCmUmU YES YES YES YESmGmGdCmUdCmGmUmUmAm GmGmUmGdCmGmUmAmUmA mU3T 140 32306mAmAmUmAmUmAdCmUmUmG YES YES YES YES mGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA3T  141 32307 mAmUmAmUmAdCmUmUmGm YES YES YES YESGdCmUdCmGmUmUmAmGmG mUmGdCmGmUmAmU3T 142 33889 mUmAmUmAdCmUmUmGmGdC YESNO YES YES mUdCmGmUmUmAmGmGmUm GdCmGmUmAmU3T 143 33890mAmUmAmUmAdCmUmUmGm YES YES YES YES GdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmA3T 144 33891 mUmAmUmAdCmUmUmGmGdC YES YES YES YESmUdCmGmUmUmAmGmGmUm GdCmGmUmAmUmA3T 145 33895 mAmUmAdCmUmUmGmGdCmU YESYES YES YES dCmGmUmUmAmGmGmUmGd CmGmUmAmUmA3T 146 33900mUmUmAdCmUmUmGmGdCmU YES YES YES YES dCmGmUmUmAmGmGmUmGd CmGmUmAmUmA3T147 33907 mGmGmAmAmUmAdCmUmUm YES YES YES YES GmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAm UmA3T

Example 36

This example demonstrates that 3′-truncated derivatives of ARC19499 havefunctional activity in the CAT assay.

ARC21383, ARC21385, ARC21387 and ARC21389 have successive singledeletions at their 3′-end relative to ARC19499 (Table 39). Thesemolecules are pegylated at their 5′-ends with a 40 kDa PEG. Thesemolecules were added to hemophilia A plasma at different concentrations(300 nM-1.2 nM) and thrombin generation was measured. ARC19499 was usedas a control in the experiment. These 3′-truncated molecules all hadactivity in the CAT assay that was similar to that observed withARC19499, with respect to both endogenous thrombin potential (ETP; FIG.145A) and peak thrombin (FIG. 145B).

This example demonstrates that ARC19499 can be truncated by removal ofthe 3′-3T and the three 3′-end core nucleotides and still retainactivity in the CAT assay that is similar to that of the parent moleculeARC19499.

TABLE 39 3′-truncated versions of ARC19499 Sequence (5′ → 3′) (mN =2′-methoxy-containing residue; dN = deoxy residue; SEQ ID nh =amine linker; PEG40K = NO: ARC # 40 kDa PEG) 148 21383PEG40KnhmGmGmAmAmUmAmUmAd CmUmUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmUmAmUm A 149 21385 PEG40KnhmGmGmAmAmUmAmUmAdCmUmUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmAmU 150 21387PEG40KnhmGmGmAmAmUmAmUmAd CmUmUmGmGdCmUdCmGmUmUmA mGmGmUmGdCmGmUmAmUmA151 21389 PEG40KnhmGmGmAmAmUmAmUmAd CmUmUmGmGdCmUdCmGmUmUmAmGmGmUmGdCmGmUmAmU

Example 37

This example describes a strategy for identifying aptamers that bind atleast in part to or otherwise interact with one or more portions oftissue factor pathway inhibitor (TFPI). For these experiments, a partialTFPI protein or peptide that comprises the region of TFPI that is to betargeted is used as a selection target. Any portion of the TFPI proteincan be used for this type of experiment. For example, a TFPI proteinthat only contains the K3 and C-terminal domains of full-length TFPI(K3-C TFPI; amino acids 182-276) is the target for selection. A nucleicacid pool is incubated with K3-C TFPI to allow binding to occur, themixture is partitioned to separate bound nucleic acids from unboundnucleic acids, and the bound nucleic acids are eluted from the proteinand amplified. This process is optionally repeated for multiple cyclesuntil an aptamer is identified. For some cycles, full-length TFPI isused as the target to ensure that the aptamer binds to the K3-C-terminalregion of the protein in the context of the full-length protein. Theseexperiments result in the identification of TFPI-binding aptamers whosebinding epitope is contained, for example, within the K3-C-terminalregion of the protein.

Example 38

This example describes a strategy for identifying aptamers that bind atleast in part to or otherwise interact with one or more portions oftissue factor pathway inhibitor (TFPI). For these experiments,full-length TFPI is used as a selection target and a portion of TFPI ora ligand that binds to TFPI is used to elute aptamers that bind to aportion of the TFPI protein. A protein or peptide that contains only aportion of TFPI could also be used as the selection target. For example,a peptide comprised of amino acids 150-190 of TFPI is used for elution.A nucleic acid pool is incubated with full-length TFPI to allow bindingto occur, the mixture is partitioned to separate bound nucleic acidsfrom unbound nucleic acids, bound nucleic acids are eluted from theprotein by incubation with the TFPI 150-190 peptide, and amplified. Thisprocess is repeated for multiple cycles until an aptamer is identified.These experiments result in the identification of TFPI-binding aptamerswhose binding epitope contains, for example, all or part of the 150-190region of TFPI.

Example 39

This example describes a strategy for identifying aptamers that bind atleast in part to or otherwise interact with one or more portions oftissue factor pathway inhibitor (TFPI). For these experiments,full-length TFPI is used as a selection target and a ligand that bindsto TFPI is included in the selection to block epitopes from aptamerbinding to drive aptamer binding to alternative sites on the protein. Aprotein or peptide that contains only a portion of TFPI could also beused as the selection target. The blocking ligand is included in theselection step and/or in the partitioning step as a capture method or asa washing reagent. For example, an antibody that binds to the C-terminusof TFPI is included in the selection. A nucleic acid pool is incubatedwith TFPI in the presence of the antibody to allow binding to occur, themixture is partitioned to separate bound nucleic acids from unboundnucleic acids, and bound nucleic acids are eluted from the protein andamplified. This process is repeated for multiple cycles until an aptameris identified. Some cycles include the antibody in the washing solutionduring the partitioning step and some cycles use the antibody as apartitioning method to capture TFPI-aptamer complexes, in conjunctionwith or instead of inclusion of the antibody in the binding step. Theseexperiments result in the identification of TFPI-binding aptamers whosebinding epitope is not contained, for example, within theantibody-binding region at the C-terminus of TFPI.

Example 40

This example describes a strategy for identifying aptamers that bind atleast in part to or otherwise interact with one or more portions oftissue factor pathway inhibitor (TFPI). For these experiments,full-length TFPI is used as a selection target and a partitioning stepis employed that separates aptamers with a desired functional propertyaway from nucleic acids that do not have that functional property. Aprotein or peptide that contains only a portion of TFPI could also beused as the selection target. For example, the desired aptamerfunctional property is inhibition of TFPI interaction with Factor Xa(FXa). For these experiments, a nucleic acid pool is incubated with TFPIunder conditions that allow for binding and then partitioned to separateunbound nucleic acids from aptamers that are bound to TFPI. TheTFPI-bound aptamers are then incubated with FXa that is bound to ahydrophobic plate. Free TFPI and TFPI bound with aptamers that do notinterfere with the TFPI-FXa interaction bind to FXa on the plate, whileTFPI bound with aptamers that do interfere with the TFPI-FXa interactiondo not bind to the plate. Aptamers from the unbound TFPI-aptamercomplexes are then amplified. This process is repeated for multiplecycles until an aptamer is identified. These experiments result, forexample, in the identification of aptamers that bind to a region of theTFPI protein that mediates inhibition of the functional interactionbetween TFPI and FXa.

Example 41

This example describes a strategy for identifying antibodies that bindat least in part to or otherwise interact with one or more portions oftissue factor pathway inhibitor (TFPI). The antigen can be any one ormore portions of TFPI that are of interest. For example, the antigen maybe a TFPI protein that only contains the K3 and C-terminal domains offull-length TFPI (K3-C TFPI; amino acids 182-276). The antigen isexpressed in an expression system or is synthesized on an automatedprotein synthesizer. Mice are then immunized with the antigen insolution. Antibody producing cells are then isolated from the immunizedmice and fused with myeloma cells to form monoclonal antibody-producinghybridomas. The hybridomas are then cultured in a selective medium. Theresulting cells are then plated by serial dilution and assayed for theproduction of antibodies that specifically bind to the antigen. Selectedmonoclonal antibody secreting hybridomas are then cultured. Antibodiesare then purified from the culture media supernatants of hybridomacells. These experiments result in the identification of TFPI-bindingantibodies whose binding epitope is contained, for example, within theK3-C-terminal region of the protein.

The invention having now been described by way of written descriptionand example, those of skill in the art will recognize that the inventioncan be practiced in a variety of embodiments and that the descriptionand examples above are for purposes of illustration and not limitationof the following claims.

1) An aptamer that binds to tissue factor pathway inhibitor (TFPI),wherein the aptamer comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs.: 4, 1, 2, 3, 5, 6, 7, 8, 9 and
 10. 2)The aptamer of claim 1, wherein the TFPI is human TFPI. 3) The aptamerof claim 1, wherein the aptamer has a dissociation constant for humanTFPI of 100 nM or less. 4) The aptamer of claim 1, wherein the aptamercomprises at least one chemical modification. 5) The aptamer of claim 4,wherein the modification is selected from the group consisting of: achemical substitution at a sugar position, a chemical substitution at aninternucleotide linkage, and a chemical substitution at a base position.6) The aptamer of claim 4, wherein the modification is selected from thegroup consisting of: incorporation of a modified nucleotide; a 3′ cap; a5′ cap; conjugation to a high molecular weight, non-immunogeniccompound; conjugation to a lipophilic compound; incorporation of a CpGmotif; and incorporation of a phosphorothioate or phosphorodithioateinto the phosphate backbone. 7) The aptamer of claim 6, wherein the highmolecular weight, non-immunogenic, compound is polyethylene glycol. 8)The aptamer of claim 6, wherein the 3′ cap is an inverted deoxythymidinecap. 9) The aptamer of claim 1 or a salt thereof comprising the nucleicacid sequence set forth below:mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA(SEQ ID NO: 1) (ARC26835), wherein “dN” is a deoxynucleotide and “mN” isa 2′-OMe containing nucleotide. 10) The aptamer of claim 1 or a saltthereof comprising the nucleic acid sequence set forth below:mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2) (ARC17480), wherein “3T” is an inverted deoxythymidine,“dN” is a deoxynucleotide and “mN” is a 2′-OMe containing nucleotide.11) The aptamer of claim 1 or a salt thereof comprising the nucleic acidsequence set forth below:NH₂-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 3) (ARC19498), wherein “NH₂” is from a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-OMe containing nucleotide. 12) Theaptamer of claim 1 or a salt thereof comprising the nucleic acidsequence set forth below:PEG40K—NH-mG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 4) (ARC19499), wherein “NH” is from a 5′-hexylamine linkerphosphoramidite, “PEG” is a polyethylene glycol, “3T” is an inverteddeoxythymidine, “dN” is a deoxynucleotide and “mN” is a 2′-OMecontaining nucleotide. 13) A pharmaceutical composition comprising atherapeutically effective amount of the aptamer of claim 1 or a saltthereof, and a pharmaceutically acceptable carrier or diluent. 14) Amethod for treating, preventing, delaying the progression orameliorating a bleeding disorder mediated by TFPI, comprisingadministering to a subject the composition of claim
 13. 15) The methodof claim 14, wherein the subject is a mammal. 16) The method of claim15, wherein the mammal is a human. 17) The method of claim 14, whereinthe bleeding disorder is selected from the group consisting of:prophylaxis and/or on-demand therapy for coagulation factordeficiencies, congenital or acquired, mild/moderate/severe, includinghemophilia A (Factor VIII-deficiency), hemophilia B (Factor IXdeficiency) and hemophilia C (Factor XI deficiency); hemophilia A or Bwith inhibitors; other factor deficiencies (V, VII, X, XIII,prothrombin, fibrinogen); deficiency of α2-plasmin inhibitor; deficiencyof plasminogen activator inhibitor 1; multiple factor deficiency;functional factor abnormalities (e.g., dysprothrombinemia); jointhemorrhage (hemarthrosis), including, but not limited to, ankle, elbowand knee; spontaneous bleeding in other locations (muscle,gastrointestinal, mouth, etc.); hemorrhagic stroke; intracranialhemorrhage; lacerations and other hemorrhage associate with trauma;acute traumatic coagulopathy; coagulopathy associated with cancer (e.g.,acute promyelocytic leukemia); von Willebrand's Disease; disseminatedintravascular coagulation; liver disease; menorrhagia; thrombocytopeniaand hemorrhage associated with the use of anticoagulants (e.g., vitaminK antagonists, FXa antagonists, etc.). 18) The method of claim 14,wherein the composition is administered prior to, during and/or after amedical procedure. 19) The method of claim 14, wherein the compositionis administered in combination with another drug. 20) The method ofclaim 14, wherein the composition is administered in combination withanother therapy. 21) A kit comprising the aptamer of claim
 1. 22) Anaptamer or a salt thereof that binds to tissue factor pathway inhibitor(TFPI), wherein the aptamer comprises the following structure:

wherein HN

PO₃H is from a 5′-amine linker phosphoramidite, and wherein the aptamercomprises the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2), wherein “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide, andwherein 20KPEG is a mPEG moiety having a molecular weight of 20 kDa. 23)The aptamer of claim 22, wherein the linker is selected from the groupconsisting of: 2-18 consecutive CH₂ groups, 2-12 consecutive CH₂ groups,4-8 consecutive CH₂ groups and 6 consecutive CH₂ groups. 24) An aptameror a salt thereof that binds to tissue factor pathway inhibitor (TFPI),wherein the aptamer comprises the following structure:

wherein the aptamer comprises the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2), wherein “3T” is an inverted deoxythymidine, “dN” is adeoxynucleotide and “mN” is a 2′-O Methyl containing nucleotide, andwherein 20KPEG is a mPEG moiety having a molecular weight of 20 kDa. 25)An aptamer or a salt thereof that binds to tissue factor pathwayinhibitor (TFPI), wherein the aptamer comprises the following structure:

wherein the aptamer comprises the nucleic acid sequence ofmG-mG-mA-mA-mU-mA-mU-mA-dC-mU-mU-mG-mG-dC-mU-dC-mG-mU-mU-mA-mG-mG-mU-mG-dC-mG-mU-mA-mU-mA-mU-mA-3T(SEQ ID NO: 2), wherein “n” is approximately 450, “3T” is an inverteddeoxythymidine, “dN” is a deoxynucleotide and “mN” is a 2′-O Methylcontaining nucleotide. 26) A reversal agent comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO: 15 (ARC23085),SEQ ID NO: 16 (ARC23087); SEQ ID NO: 17 (ARC23088) and SEQ ID NO: 18(ARC23089). 27) An aptamer that binds to a human tissue factor pathwayinhibitor (TFPI) polypeptide having the amino acid sequence of SEQ IDNO: 11, wherein the aptamer modulates TFPI-mediated inhibition of bloodcoagulation, and wherein the aptamer competes for binding to TFPI with areference aptamer comprising a nucleic acid sequence selected from thegroup consisting of: SEQ ID NO: 4 (ARC19499), SEQ ID NO: 1 (ARC26835),SEQ ID NO: 2 (ARC17480), SEQ ID NO: 3 (ARC19498), SEQ ID NO: 5(ARC19500), SEQ ID NO:6 (ARC19501), SEQ ID NO: 7 (ARC31301), SEQ ID NO:8 (ARC18546), SEQ ID NO: 9 (ARC19881) and SEQ ID NO: 10 (ARC19882). 28)An aptamer that binds to a region on a human tissue factor pathwayinhibitor (TFPI) polypeptide comprising one or more portions of SEQ IDNO: 11, wherein the one or more portions is selected from the groupconsisting of: amino acid residues 148-170, amino acid residues 150-170,amino acid residues 155-175, amino acid residues 160-180, amino acidresidues 165-185, amino acid residues 170-190, amino acid residues175-195, amino acid residues 180-200, amino acid residues 185-205, aminoacid residues 190-210, amino acid residues 195-215, amino acid residues200-220, amino acid residues 205-225, amino acid residues 210-230, aminoacid residues 215-235, amino acid residues 220-240, amino acid residues225-245, amino acid residues 230-250, amino acid residues 235-255, aminoacid residues 240-260, amino acid residues 245-265, amino acid residues250-270, amino acid residues 255-275, amino acid residues 260-276, aminoacid residues 148-175, amino acid residues 150-175, amino acid residues150-180, amino acid residues 150-185, amino acid residues 150-190, aminoacid residues 150-195, amino acid residues 150-200, amino acid residues150-205, amino acid residues 150-210, amino acid residues 150-215, aminoacid residues 150-220, amino acid residues 150-225, amino acid residues150-230, amino acid residues 150-235, amino acid residues 150-240, aminoacid residues 150-245, amino acid residues 150-250, amino acid residues150-255, amino acid residues 150-260, amino acid residues 150-265, aminoacid residues 150-270, amino acid residues 150-275, amino acid residues150-276, amino acid residues 190-240, amino acid residues 190-276, aminoacid residues 240-276, amino acid residues 242-276, amino acid residues161-181, amino acid residues 162-181, amino acid residues 182-240, aminoacid residues 182-241, and amino acid residues 182-276. 29) An aptamerthat binds to human tissue factor pathway inhibitor (TFPI) and exhibitsone or more of the following properties: a) competes for binding to TFPIwith any one of SEQ ID NOs: 1-10; b) inhibits TFPI inhibition of FactorXa; c) increases thrombin generation in hemophilia plasma; d) inhibitsTFPI inhibition of the intrinsic tenase complex; e) restores normalhemostasis, as measured by thromboelastography (TEG) in whole blood andplasma; f) restores normal clotting, as indicated by shorter clot time,more rapid clot formation or more stable clot development, as measuredby thromboelastography (TEG) or rotational thromboelastometry (ROTEM) inwhole blood and plasma; or g) decreases the clot time, as measured bydilute prothrombin time (dPT), tissue factor activated clotting time(TF-ACT) or any other TFPI-sensitive clot-time measurement. 30) Anaptamer that binds to human tissue factor pathway inhibitor wherein theaptamer competes for binding to TFPI with a reference aptamer selectedfrom the group consisting of: SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9 and SEQ ID NO:
 10. 31) An aptamer that binds to tissuefactor pathway inhibitor (TFPI) wherein the aptamer competes, eitherdirectly or indirectly, for binding to TFPI with a reference antibodyselected from the group consisting of: AD4903. 32) An aptamer that bindsto human tissue factor pathway inhibitor (TFPI) and comprises a stem andloop motif having the nucleotide sequence of SEQ ID NO: 4, wherein: a)any one or more of nucleotides 1, 2, 3, 4, 6, 8, 11, 12, 13, 17, 20, 21,22, 24, 28, 30 and 32 may be modified from a 2′-OMe substitution to a2′-deoxy substitution; b) any one or more of nucleotides 5, 7, 15, 19,23, 27, 29 and 31 may be modified from a 2′-OMe uracil to either a2′-deoxy uracil or a 2′-deoxy thymine; c) nucleotide 18 may be modifiedfrom a 2′-OMe uracil to a 2′-deoxy uracil; and/or d) any one or more ofnucleotides 14, 16 and 25 may be modified from a 2′-deoxy cytosine toeither a 2′-OMe cytosine or a 2′-fluoro cytosine. 33) An aptamer thatbinds to tissue factor pathway inhibitor (TFPI), wherein the aptamercomprises a primary nucleic acid sequence selected from the groupconsisting of SEQ ID NOs.: 4, 1, 2, 3, 5, 6, 7, 8, 9 and 10.